Adhesive tissue repair patch

ABSTRACT

A method of tissue repair is provided using a biocompatible nonimmunogenic adhesive composition. The adhesive composition comprises collagen and a plurality of crosslinkable components having reactive functional groups thereon, with the functional groups selected so as to enable inter-reaction between the components, i.e., crosslinking. Kits for use in carrying out the method of the invention are also provided, as are pretreated surgically acceptable patches that have been coated with the aforementioned adhesive composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/262,640filed Sep. 30, 2002, now U.S. Pat. No. 6,833,408, now allowed, which isa continuation-in-part of U.S. application Ser. No. 09/883,138 filedJun. 15, 2001, now U.S. Pat. No. 6,458,889, which is acontinuation-in-part of U.S. application Ser. No. 09/733,739 filed Dec.8, 2000, now U.S. Pat. No. 6,323,278, which is a continuation of U.S.application Ser. No. 09/302,852 filed Apr. 30, 1999, now U.S. Pat. No.6,166,130, which was a continuation of U.S. application Ser. No.09/229,851 filed Jan. 13, 1999, now U.S. Pat. No. 6,051,648, which was acontinuation of U.S. application Ser. No. 08/769,806 filed Dec. 18,1996, now U.S. Pat. No. 5,874,500, which was a continuation-in-part ofU.S. application Ser. No. 08/573,799, filed Dec. 18, 1995, nowabandoned. This application is also a continuation-in-part of U.S.application Ser. No. 09/649,337 filed Aug. 28, 2000, now U.S. Pat. No.6,495,127, claiming priority to U.S. Provisional Application Ser. No.60/151,273, filed Aug. 27, 1999. The disclosures of the aforementionedapplications are incorporated by reference in their entireties.

TECHNICAL FIELD

This invention relates generally to methods for repairing tissue usingadhesive compositions comprised of a hydrophilic polymer and crosslinkedbiomaterials. More specifically, the invention relates to tissue repairusing a composition of collagen and crosslinkable components.

BACKGROUND OF THE INVENTION

Tissue damage can result from many causes. Examples of such causesinclude surgical incisions, such as internal and epidermal surgicalincisions; prosthetic implants, including injury attendant surgery suchas hip replacements; and wounds, including lacerations, incisions, andpenetrations. Often such damage is the result of herniation wherein theouter layers of the abdominal wall weaken, tear, or bulge. The resultingweakened area or hole allows for sections of the inner lining of theabdominal cavity, or peritoneum, to protrude. This protrusion can bepainful and if uncorrected can result in strangulation of the protrudingtissue. Although almost all tissue can become herniated, the tissue inthe inguinal canal, in the navel, and surrounding the location of formerincisions are most common. Since the early 1980's, the surgicaltechniques used in repairing inguinal or groin hernias have undergone aprofound transformation. One such technique incorporates a surgicallyacceptable patch as part of the groin hernia repair. Goussous. (1995),“Effectiveness of The Mesh Plug Technique” (letter). Surgery; 117:600.Over time scar tissue forms around the reinforcing mesh, creating asupporting wall to minimize future hernias.

Various methods for approaching the herniated tissue and affixing themesh prosthesis have been developed. There are two primary techniquesused in hernia repair. In the traditional “open surgery” technique, thesurgeon makes a three- to four-inch incision in the abdominal wall,pushes the hernial sac inside and uses mesh to reinforce the abdominalwall. The other method of hernia repair is the laparoscopic technique,wherein three tiny incisions, about the size of dime, provide thesurgeon sufficient access to reposition the hernia sac back through itshole and secure a mesh patch over the weak area in the muscle wall. Theincisions used in the laparoscopic technique are sufficiently small sothat they can be covered by adhesive strips and there is minimal or noscarring.

The surgically acceptable patch used in both of the above-discussedtechniques is generally held in place via suturing or stapling to thesurrounding tissue. Unfortunately, the use of such sutures or staplesmay increase the patient's discomfort and increase the incidences ofwound infection, vascular injury and entrapment neuropathy. Whileherniorrhaphies have been conducted without firmly connecting the patchto the tissue surface and allowing the pressure of the peritoneum tohold the patch against the posterior side of the abdominal wall, seeZieren et al. (1999) “Is Mesh Fixation Necessary in Abdominal HerniaRepair?” Lang. Arch Surg. 384:71-75, fixation of the patch is generallypreferred in order to avoid folding, shrinkage, and migration of thepatch and is usually considered to be essential in laparoscopicprocedures.

Recently cyanoacrylates and fibrin glues have been used as fixatives inhernia repair. While Katkhouda et al. (2001) Ann. Surg 233:18-25 presentthe use of a fibrin sealant as a patch fixative, such fibrin productsare made from human products and are thus susceptible to contamination.Also, fibrin adhesives are difficult to prepare and to store. The use ofcyanoacrylates as adhesives also presents problems in that the adhesivemay not be biocompatible and may not provide a sufficient degree ofelasticity thereby resulting in increased patent discomfort and anincreased incidence of reoccurrence. See, Farouk et al. (1996),“Preliminary Experience with utyl-2-Cyanoacrylate adhesive inTension-Free Inguinal Hernia Repair,” Brit. J. Surg. 83:1100 and Jourdanet al. (1998), “The Use of N-Butyl-2-Cyanoacrylate Glue for the Fixationof Polypropylene Mesh in Laparoscopic Hernia Repair,” 6^(th) World Congof Endo. Surg., 1221-1225.

A new method of tissue repair has now been developed using a surgicallyacceptable hydrophilic-based crosslinking adhesive. The use of thisadhesive composition avoids the potential complications inherent insuture or staple based methods of tissue attachment. Also, as thehydrophilic polymer-based adhesive does not contain human bloodproducts, the danger of contamination present with fibrin adhesives isremoved. While providing a stronger adhesive bond than fibrin adhesivessuch as TISSEEL®, the hydrophilic polymer-based crosslinking adhesive ismuch more flexible than cyanoacrylate adhesives and is completelybiocompatible.

U.S. Pat. No. 5,162,430, to Rhee et al., and commonly owned by theassignee of the present invention, discloses collagen-synthetic polymerconjugates prepared by covalently binding collagen to synthetichydrophilic polymers such as various derivatives of polyethylene glycol.

Commonly owned U.S. Pat. No. 5,324,775, to Rhee et al., disclosesvarious inert, naturally occurring, biocompatible polymers (such aspolysaccharides) covalently bound to synthetic, hydrophilic polyethyleneglycol polymers.

Commonly owned U.S. Pat. No. 5,328,955, to Rhee et al., disclosesvarious activated forms of polyethylene glycol and various linkageswhich can be used to produce collagen-synthetic polymer conjugateshaving a range of physical and chemical properties.

Commonly owned, U.S. application Ser. No. 08/403,358, filed Mar. 14,1995, now abandoned, a European counterpart of which was published as EP96102366, discloses a crosslinked biomaterial composition that isprepared using a hydrophobic crosslinking agent, or a mixture ofhydrophilic and hydrophobic crosslinking agents. Preferred hydrophobiccrosslinking agents include any hydrophobic polymer that contains, orcan be chemically derivatized to contain, two or more succinimidylgroups.

Commonly owned U.S. Pat. No. 5,580,923 to Yeung et al., discloses acomposition useful in the prevention of surgical adhesions. Thecomposition has a substrate, which is preferably collagen and a bindingagent, which preferably has at least one tissue-reactive functionalgroup and at least one substrate-reactive functional group.

Commonly owned U.S. Pat. No. 5,614,587 to Rhee et al., disclosesbioadhesive compositions having collagen crosslinked using amultifunctionally activated synthetic hydrophilic polymer, as well asmethods of using such compositions to effect adhesion between a firstsurface and a second surface. At least one of the first and secondsurfaces is preferably a native tissue surface.

Japanese patent publication No. 07090241 discloses a composition usedfor temporary adhesion of a lens material to a machining device, whichcontains a mixture of polyethylene glycol, having an average molecularweight in the range of 1000-5000, and poly-N-vinylpyrrolidone, having anaverage molecular weight in the range of 30,000-200,000.

West and Hubbell, Biomaterials (1995) 16:1153-1156, disclose theprevention of post-operative adhesions using a photopolymerizedpolyethylene glycol-co-lactic acid diacrylate hydrogel and a physicallycrosslinked polyethylene glycol-co-polypropylene glycol hydrogel,Poloxamer 407®.

Each publication cited above and is incorporated herein by reference todescribe and disclose the subject matter for which it is cited.

The invention is directed to a method of repairing tissue, such asherniated tissue, using a versatile biocompatible adhesive compositionnot previously disclosed or envisioned by those in the biomaterialfield. The composition has a hydrophilic polymer and crosslinkablecomponents that may be readily crosslinked upon admixture with anaqueous medium to provide a crosslinked composition suitable for use asa bioadhesive. The adhesive composition is biocompatible, and does notleave any toxic, inflammatory or immunogenic reaction products at thesite of administration. Preferably, the composition is not subject toenzymatic cleavage by matrix metalloproteinases such as collagenase, andis therefore not readily degradable in vivo. As a result, the adhesivecomposition will degrade more slowly than either the hydrophilic polymercomponent or the crosslinkable component as the two components willserve to mutually protect each other from the effects ofmetalloproteases or hydrolysis.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the invention, a method for tissue repairis provided utilizing a readily crosslinkable, biocompatible, adhesivecomposition to secure a surgically acceptable patch to the damagedtissue. The adhesive composition is comprised of a hydrophilic polymer,a crosslinkable component A having m nucleophilic groups, wherein m≧2;and a crosslinkable component B having n electrophilic groups capable ofreaction with the m nucleophilic groups to form covalent bonds, whereinn≧2 and m+n≧4. In the composition, each of components A and B isbiocompatible and nonimmunogenic, at least one of components A and B isa hydrophilic polymer, and admixture of components A and B in an aqueousmedium results in crosslinking of the composition to give abiocompatible, nonimmunogenic, crosslinked matrix.

Each of the crosslinkable components may be polymeric, in which case atleast two crosslinkable components are generally although notnecessarily composed of a purely synthetic polymer rather than anaturally occurring or semi-synthetic polymer, wherein“semi-synthetic”refers to a chemically modified naturally occurringpolymer. Alternatively, one or two of crosslinkable components A and Bmay be a low molecular weight crosslinking agent, typically an agentcomprised of a hydrocarbyl moiety containing 2 to 14 carbon atoms and atleast two functional groups, i.e., nucleophilic or electrophilic groups,depending on the component. For convenience, the term “polynucleophilic”will be used herein to refer to a compound having two or morenucleophilic moieties, and the term “polyelectrophilic” will be used torefer to a compound having two or more electrophilic moieties. Theadhesive composition may also additionally comprise an optional thirdbiocompatible and nonimmunogenic crosslinkable component C having atleast one functional group selected from (i) nucleophilic groups capableof reacting with the electrophilic groups of component B and (ii)electrophilic groups capable of reacting with the nucleophilic groups ofcomponent A.

Any conventional surgical procedure may be used to access the herniatedtissue and any conventional surgically acceptable patch may be affixedwith the adhesive composition. For example, a polypropylene patch may beaffixed to the posterior surface of the abdominal wall via laparoscopicsurgical techniques or may be affixed to the anterior surface via opensurgical techniques. The method is applicable to a wide variety ofhernia types, including but not limited to, inguinal hernias, femoralhernias, scrotal hernias, ventral hernias, umbilical hernias,ventral/epigastric hernias, incisional hernias, spigelian hernias,recurrent hernias, recurrent incisional hernias, bilateral hernias,stoma hernias, and hiatus hernias.

In another aspect of the invention, a kit is provided comprising theadhesive composition as discussed above and a surgically acceptablepatch.

In a still further aspect of the invention, a pretreated patch isprovided comprising a surgically acceptable patch that has been coatedwith the adhesive composition as discussed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 10 schematically illustrate reaction of variouspolyelectrophilic components with substituted polyethylene glycol (PEG)as a representative polynucleophile. In FIGS. 1-10, thepolyelectrophilic components are composed of a pentaerythritol core witheach of the four hydroxyl groups substituted with PEG, and with each PEGbranch terminated with a reactive electrophilic group.

FIG. 11 presents the results of a comparative peel strength test for anadhesive composition of the invention in a 2×2 cm polypropylenemesh/collagen membrane test.

FIG. 12 graphically illustrates the results of peel strength testing forvarious adhesives when used to adhere lab grade polypropylene mesh to acollagen membrane surface as described in Example 3.

FIG. 13 graphically illustrates the changes in peel strength over a 24hour period when an adhesive of the invention was used to affix labgrade polypropylene mesh to a cowhide strip as described in Example 4.

FIG. 14 graphically illustrates the results of an adhesive compositioncomprised of pentaerythritol polyethylene glycol ethertetra-succinimidyl glutarate, pentaerythritol polyethylene glycol ethertetra-sulfhydryl, and methylated collagen, an adhesive compositioncomprised of pentaerythritol polyethylene glycol ethertetra-succinimidyl glutarate, pentaerythritol polyethylene glycol ethertetra-sulfhydryl, poly(L-lactic acid) fiber, and methylated collagen,KRAZY GLUE® (Toagosei Co., Ltd., Tokyo Japan) and TISSEEL® (Immuno,Aktiengesellschaft fur Chemischmedizinische Produkte, Postfach Austria)when used to affix a 2×2 cm polypropylene mesh strip onto a cowhidestrip.

FIG. 15 graphically illustrates the results of comparative pull strengthtesting for an adhesive composition comprised of pentaerythritolpolyethylene glycol ether tetra-succinimidyl glutarate, pentaerythritolpolyethylene glycol ether tetra-sulfhydryl, and methylated collagen andan adhesive composition comprised of pentaerythritol polyethylene glycolether tetra-succinimidyl glutarate, pentaerythritol polyethylene glycolether tetra-sulfhydryl, poly(L-lactic acid) fiber, and methylatedcollagen when used to affix a 2×2 cm patch of BARD® (C.R. BARD, INC.,Murray Hill, N.J.) polypropylene mesh to a cowhide strip as described inExample 6.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Nomenclature

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto particular compositional forms, crosslinkable components,crosslinking techniques, or methods of use, as such may vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example, “acrosslinkable component” refers not only to a single crosslinkablecomponent but also to a combination of two or more differentcrosslinkable components, “a hydrophilic polymer” refers to acombination of hydrophilic polymers as well as to a single hydrophilicpolymer, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Although any methods and materialssimilar or equivalent to those described herein may be useful in thepractice or testing of the present invention, preferred methods andmaterials are described below. All patents, patent applications andother publications mentioned herein are incorporated herein byreference. Specific terminology of particular importance to thedescription of the present invention is defined below.

As used herein, the terms “bioadhesive”, “biological adhesive”, and“surgical adhesive” are used interchangeably to refer to biocompatiblecompositions capable of effecting temporary or permanent attachmentbetween the surfaces of two native tissues, or between a native tissuesurface and either a non-native tissue surface or a surface of asynthetic implant.

The term “surgically acceptable” refers to those items, e.g. patches,that are biocompatible, and are otherwise acceptable for surgical use.

The term “crosslinked” herein refers to a composition containingintermolecular crosslinks and optionally intramolecular crosslinks aswell, arising from the formation of covalent bonds. Covalent bondingbetween two crosslinkable components may be direct, in which case anatom in one component is directly bound to an atom in the othercomponent, or it may be indirect, through a linking group. A crosslinkedmatrix may, in addition to covalent bonds, also include intermolecularand/or intramolecular noncovalent bonds such as hydrogen bonds andelectrostatic (ionic) bonds. The term “crosslinkable” refers to acomponent or compound that is capable of undergoing reaction to form acrosslinked composition.

The terms “nucleophile” and “nucleophilic” refer to a functional groupthat is electron rich, has an unshared pair of electrons acting as areactive site, and reacts with a positively charged orelectron-deficient site, generally present on another molecule.

The terms “electrophile” and “electrophilic” refer to a functional groupthat is susceptible to nucleophilic attack, i.e., susceptible toreaction with an incoming nucleophilic group. Electrophilic groupsherein are positively charged or electron-deficient, typicallyelectron-deficient.

The term “activated” refers to a modification of an existing functionalgroup to generate or introduce a new reactive functional group from theprior existing functional group, wherein the new reactive functionalgroup is capable of undergoing reaction with another functional group toform a covalent bond. For example, a component containing carboxylicacid (—COOH) groups can be activated by reaction withN-hydroxysuccinimide or N-hydroxysulfo-succinimide using knownprocedures, to form an activated carboxylate (which is a reactiveelectrophilic group), i.e., an N-hydroxysuccinimide ester or anN-hydroxysulfosuccinimide ester, respectively. In another example,carboxylic acid groups can be activated by reaction with an acyl halide,e.g., an acyl chloride, again using known procedures, to provide anactivated electrophilic group in the form of an anhydride.

The terms “hydrophilic” and “hydrophobic” are generally defined in termsof a partition coefficient P, which is the ratio of the equilibriumconcentration of a compound in an organic phase to that in an aqueousphase. A hydrophilic compound has a log P value less than 1.0, typicallyless than about −0.5, where P is the partition coefficient of thecompound between octanol and water, while hydrophobic compounds willgenerally have a log P greater than about 3.0, typically greater thanabout 5.0. Preferred crosslinkable components herein are hydrophilic,although as long as the crosslinkable composition as a whole contains atleast one hydrophilic component, crosslinkable hydrophobic componentsmay also be present.

The term “polymer” is used not only in the conventional sense to referto molecules composed of repeating monomer units, includinghomopolymers, block copolymers, random copolymers, and graft copolymers,but also, as indicated in parent application Ser. No. 09/733,739, nowU.S. Pat. No. 6,323,728, to refer to polyfunctional small molecules thatdo not contain repeating monomer units but are “polymeric” in the senseof being “polyfunctional,” i.e., containing two or more functionalgroups. Accordingly, it will be appreciated that when the term “polymer”is used, difunctional and polyfunctional small molecules are included.Such moieties include, by way of example: the difunctional electrophilesdisuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidylpropionate) (DSP),bis(2-succinimidooxy-carbonyloxy) ethyl sulfone (BSOCOES),3,3′-dithiobis(sulfosuccinimidylpropionate (DTSSP); and the di- andpolyfunctional nucleophiles ethylenediamine (H₂N—CH₂—CH₂—NH₂),tetramethylene diamine (H₂N-[CH₂]₄—NH₂), pentamethylene diamine(cadaverine) (H₂N-[CH₂]₅—NH₂), hexamethylene diamine (H₂N-[CH₂]₆—NH₂),bos(2-aminoethyl)amine (HN—[CH₂—CH₂—NH₂]₂), and tris (2-aminoethyl)amine(N-[CH₂—CH₂—NH₂]₃). All suitable polymers herein are nontoxic,non-inflammatory and nonimmunogenic, and will preferably be essentiallynondegradable in vivo over a period of up to 30 days in vivo.

The term “synthetic” to refer to various polymers herein is intended tomean “chemically synthesized.” Therefore, a synthetic polymer in thepresent compositions may have a molecular structure that is identical toa naturally occurring polymer, but the polymer per se, as incorporatedin the compositions of the invention, has been chemically synthesized inthe laboratory or industrially. “Synthetic” polymers also includesemi-synthetic polymers, i.e., naturally occurring polymers, obtainedfrom a natural source, that have been chemically modified in some way.Generally, however, the synthetic polymers herein are purely synthetic,i.e., they are neither semi-synthetic nor have a structure that isidentical to that of a naturally occurring polymer.

The term “synthetic hydrophilic polymer” as used herein refers to asynthetic polymer composed of molecular segments that render the polymeras a whole “hydrophilic,” as defined above. Preferred polymers arehighly pure or are purified to a highly pure state such that the polymeris or is treated to become pharmaceutically pure. Most hydrophilicpolymers can be rendered water soluble by incorporating a sufficientnumber of oxygen (or less frequently nitrogen) atoms available forforming hydrogen bonds in aqueous solutions. Hydrophilic polymers usefulherein include, but are not limited to: polyalkylene oxides,particularly polyethylene glycol and poly(ethylene oxide)-poly(propyleneoxide) copolymers, including block and random copolymers; polyols suchas glycerol, polyglycerol (particularly highly branched polyglycerol),propylene glycol and trimethylene glycol substituted with one or morepolyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated glycerol,mono- and di-polyoxy-ethylated propylene glycol, and mono- anddi-polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,polyoxyethylated glucose; acrylic acid polymers and analogs andcopolymers thereof, such as polyacrylic acid per se, polymethacrylicacid, poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxideacrylate) and copolymers of any of the foregoing, and/or with additionalacrylate species such as aminoethyl acrylate and mono-2-(acryloxy)-ethylsuccinate; polymaleic acid; poly(acrylamides) such as polyacrylamide perse, poly(methacrylamide), poly(dimethylacrylamide), andpoly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as poly(vinylalcohol); poly(N-vinyl lactams) such as poly(vinyl pyrrolidone),poly(N-vinyl caprolactam), and copolymers thereof, polyoxazolines,including poly(methyloxazoline) and poly(ethyloxazoline); andpolyvinylamines.

Hydrophobic polymers, including low molecular weight polyfunctionalspecies, can also be used in the crosslinkable compositions of theinvention. Hydrophobic polymers preferably contain, or can bederivatized to contain, two or more electrophilic groups, such assuccinimidyl groups, most preferably, two, three, or four electrophilicgroups. Generally, “hydrophobic polymers” herein contain a relativelysmall proportion of oxygen and/or nitrogen atoms. Preferred hydrophobicpolymers for use in the invention generally have a carbon chain that isno longer than about 14 carbons. Polymers having carbon chainssubstantially longer than 14 carbons generally have very poor solubilityin aqueous solutions and, as such, have very long reaction times whenmixed with aqueous solutions of synthetic polymers containing multiplenucleophilic groups.

The term “collagen” as used herein refers to all forms of collagen,including those, which have been processed or otherwise modified.Preferred collagens do not posses telopeptide regions (“atelopeptidecollagen”), are soluble, and may be in fibrillar or non-fibrillar form.Type I collagen is best suited to most applications involving bone orcartilage repair. However, other forms of collagen are also useful inthe practice of the invention, and are not excluded from considerationhere. Collagen crosslinked using heat, radiation, or chemical agentssuch as glutaraldehyde may also be used to form particularly rigidcrosslinked compositions. Collagen used in connection with the preferredembodiments of the invention is in a pharmaceutically pure form suchthat it can be incorporated into a human body for the intended purpose.

Those of ordinary skill in the art will appreciate that syntheticpolymers such as polyethylene glycol cannot be prepared practically tohave exact molecular weights, and that the term “molecular weight” asused herein refers to the weight average molecular weight of a number ofmolecules in any given sample, as commonly used in the art. Thus, asample of PEG 2,000 might contain a statistical mixture of polymermolecules ranging in weight from, for example, 1,500 to 2,500 daltonswith one molecule differing slightly from the next over a range.Specification of a range of molecular weights indicates that the averagemolecular weight may be any value between the limits specified, and mayinclude molecules outside those limits. Thus, a molecular weight rangeof about 800 to about 20,000 indicates an average molecular weight of atleast about 800, ranging up to about 20 kDa.

The term “cytokine” is used to describe biologically active moleculesincluding growth factors and active peptides, which aid in healing orregrowth of normal tissue. The function of cytokines is two-fold: 1)they can incite local cells to produce new collagen or tissue, or 2)they can attract cells to the site in need of correction. As such,cytokines serve to encourage “biological anchoring” of the collagenimplant within the host tissue. As previously described, the cytokinescan either be admixed with the collagen-polymer conjugate or chemicallycoupled to the conjugate. For example, one 30 may incorporate cytokinessuch as epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β (including any combination of TGF-βs), TGF-β1, TGF-β2,platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB), acidicfibroblast growth factor (FGF), basic FGF, connective tissue activatingpeptides (CTAP), β-thromboglobulin, insulin-like growth factors, tumornecrosis factors (TNF), interleukins, colony stimulating factors (CSFs),erythropoietin (EPO), nerve growth factor (NGF), interferons (IFN) bonemorphogenic protein (BMP), osteogenic factors, and the like.Incorporation of cytokines, and appropriate combinations of cytokinescan facilitate the regrowth and remodeling of the implant into normalbone tissue, or may be used in the treatment of wounds.

The term “effective amount” refers to the amount of composition requiredin order to obtain the effect desired. Thus, a “tissue growth-promotingamount” of a composition refers to the amount needed in order tostimulate tissue growth to a detectable degree. Tissue, in this context,includes connective tissue, bone, cartilage, epidermis and dermis,blood, and other tissues. The actual amount that is determined to be aneffective amount will vary depending on factors such as the size,condition, sex and age of the patient and can be more readily determinedby the caregiver.

The term “suitable fibrous material” as used herein, refers to a fibrousmaterial which is substantially insoluble in water, non-immunogenic,biocompatible, and immiscible with the crosslinkable compositions of theinvention. The fibrous material may comprise any of a variety ofmaterials having these characteristics and may be combined withcrosslinkable compositions herein in order to form and/or providestructural integrity to various implants or devices used in connectionwith medical and pharmaceutical uses. For example, the crosslinkablecompositions of the invention can be coated on the “suitable fibrousmaterial,” which can then be wrapped around a bone to provide structuralintegrity to the bone. Thus, the “suitable fibrous material” is usefulin forming the “solid implants” of the invention.

The term “in situ” as used herein means at the site of administration.Thus, the injectable reaction mixture compositions are injected orotherwise applied to a specific site within a patient's body, e.g., thelocus of the herniated tissue, and allowed to crosslink at the site ofinjection.

The term “aqueous medium” includes solutions, suspensions, dispersions,colloids, and the like containing water.

The term “substantially immediately” means within less than fiveminutes, preferably within less than two minutes, and the term“immediately” means within less than one minute, preferably within lessthan 30 seconds.

The terms “active agent,” and “biologically active agent” are usedinterchangeably herein to refer to a chemical material or compoundsuitable for administration to a patient and that induces a desiredeffect. The terms include agents that are therapeutically effective aswell as prophylactically effective. Also included are derivatives andanalogs of those compounds or classes of compounds specificallymentioned that also induce the desired effect.

The term “hydrogel” is used in the conventional sense to refer towater-swellable polymeric matrices that can absorb a substantial amountof water to form elastic gels, wherein “matrices” are three-dimensionalnetworks of macromolecules held together by covalent or noncovalentcrosslinks. Upon placement in an aqueous environment, dry hydrogelsswell to the extent allowed by the degree of cross-linking.

With regard to nomenclature pertinent to molecular structures, thefollowing definitions apply:

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group typically although not necessarilycontaining 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, aswell as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.Generally, although again not necessarily, alkyl groups herein contain 1to about 12 carbon atoms. The term “lower alkyl” intends an alkyl groupof one to six carbon atoms, preferably one to four carbon atoms.“Substituted alkyl” refers to alkyl substituted with one or moresubstituent groups. “Alkylene,” “lower alkylene” and “substitutedalkylene” refer to divalent alkyl, lower alkyl, and substituted alkylgroups, respectively.

The term “aryl” as used herein, and unless otherwise specified, refersto an aromatic substituent containing a single aromatic ring or multiplearomatic rings that are fused together, linked covalently, or linked toa common group such as a methylene or ethylene moiety. The commonlinking group may also be a carbonyl as in benzophenone, an oxygen atomas in diphenylether, or a nitrogen atom as in diphenylamine. Preferredaryl groups contain one aromatic ring or two fused or linked aromaticrings, e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine,benzophenone, and the like. “Substituted aryl” refers to an aryl moietysubstituted with one or more substituent groups, and the terms“heteroatom-containing aryl” and “heteroaryl” refer to aryl in which atleast one carbon atom is replaced with a heteroatom. The terms “arylene”and “substituted arylene” refer to divalent aryl and substituted arylgroups as just defined.

The term “heteroatom-containing” as in a “heteroatom-containinghydrocarbyl group” refers to a molecule or molecular fragment in whichone or more carbon atoms is replaced with an atom other than carbon,e.g., nitrogen, oxygen, sulfur, phosphorus or silicon.

“Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 toabout 30 carbon atoms, preferably 1 to about 24 carbon atoms, mostpreferably 1 to about 12 carbon atoms, including branched or unbranched,saturated or unsaturated species, such as alkyl groups, alkenyl groups,aryl groups, and the like. The term “lower hydrocarbyl” intends ahydrocarbyl group of one to six carbon atoms, preferably one to fourcarbon atoms. The term “hydrocarbylene” intends a divalent hydrocarbylmoiety containing 1 to about 30 carbon atoms, preferably 1 to about 24carbon atoms, most preferably 1 to about 12 carbon atoms, includingbranched or unbranched, saturated or unsaturated species, or the like.The term “lower hydrocarbylene” intends a hydrocarbylene group of one tosix carbon atoms, preferably one to four carbon atoms. “Substitutedhydrocarbyl” refers to hydrocarbyl substituted with one or moresubstituent groups, and the terms “heteroatom-containing hydrocarbyl”and “heterohydrocarbyl” refer to hydrocarbyl in which at least onecarbon atom is replaced with a heteroatom. Similarly, “substitutedhydrocarbylene” refers to hydrocarbylene substituted with one or moresubstituent groups, and the terms “heteroatom-containing hydrocarbylene”and “heterohydrocarbylene” refer to hydrocarbylene in which at least onecarbon atom is replaced with a heteroatom. If not otherwise indicated,“hydrocarbyl” indicates unsubstituted hydrocarbyl, substitutedhydrocarbyl, heteroatom-containing hydrocarbyl, and substitutedheteroatom-containing hydrocarbyl. Unless otherwise indicated, the terms“hydrocarbyl” and “hydrocarbylene” include substituted hydrocarbyl andsubstituted hydrocarbylene, heteroatom-containing hydrocarbyl andheteroatom-containing hydrocarbylene, and substitutedheteroatom-containing hydrocarbyl and substituted heteroatom-containinghydrocarbylene, respectively.

By “substituted” as in “substituted hydrocarbyl,” “substituted alkyl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the hydrocarbyl, alkyl, or other moiety, at least onehydrogen atom bound to a carbon atom is replaced with one or moresubstituents that are functional groups such as alkoxy, hydroxy, halo,nitro, and the like. Unless otherwise indicated, it is to be understoodthat specified molecular segments can be substituted with one or moresubstituents that do not compromise a compound's utility. For example,“succinimidyl” is intended to include unsubstituted succinimidyl as wellas sulfosuccinimidyl and other succinimidyl groups substituted on a ringcarbon atom, e.g., with alkoxy substituents, polyether substituents, orthe like.

Repair of Damaged Tissue

While the adhesive composition taught herein may be used in any numberof tissue repair applications, such as, but not limited to, seroma andhematoma prevention, skin and muscle flap attachment, repair andprevention of endoleaks, aortic dissection repair, lung volumereduction, neural tube repair and the making of microvasuclar and neuralanastomoses, the focus of the invention is the use of the adhesivecomposition in the repair of damaged tissue.

In the method of the invention, the repair of damaged tissue may becarried out within the context of any standard surgical process allowingaccess to and repair of the tissue, including open surgery andlaparoscopic techniques. Once the damaged tissue is accessed, theadhesive composition of the invention is placed in contact with thedamaged tissue along with any surgically acceptable patch or implant, ifneeded. When used to repair lacerated or separated tissue, such as byjoining two or more tissue surfaces, the adhesive composition is appliedto one or more of the tissue surfaces and then the surfaces are placedin contact with each other and adhesion occurs therebetween.

When used to repair herniated tissue, a surgically acceptable patch canbe attached to the area of tissue surrounding the herniated tissue so asto cover the herniated area, thereby reinforcing the damaged tissue andrepairing the defect. When attaching the patch to the surroundingtissue, the adhesive composition may be applied to either the patch, tothe surrounding tissue, or to the patch after the patch has been placedon the herniated tissue. Once the patch and tissue are brought intocontact with each other, adhesion occurs therebetween.

Preferably, all reactive components of the adhesive composition arefirst mixed to initiate crosslinking, then delivered to the desiredtissue or surface before substantial crosslinking has occurred. Thesurface or tissue to which the adhesive composition has been applied isthen contacted with the remaining surface, i.e. another tissue surfaceor implant surface, preferably immediately, to effect adhesion.

The surfaces to be adhered may be held together manually, or using otherappropriate means, while the crosslinking reaction is proceeding tocompletion. Crosslinking is typically sufficiently complete for adhesionto occur within about 5 to 60 seconds after mixing the components of theadhesive composition. However, the time required for completecrosslinking to occur is dependent on a number of factors, including thetype and molecular weight of each reactive component, the degree offunctionalization, and the concentration of the components in thecrosslinkable compositions (e.g., higher component concentrations resultin faster crosslinking times).

Surgically Acceptable Patches

When the adhesive composition of the invention is used to repair damagedtissue, it may be used in conjunction with a surgically acceptablepatch. The surgically acceptable patch may be selected from anyconventional patch type that is suitable for use in hernia repair. Manytype of patches are currently available and will be well know to one ofskill in the art. Exemplary patch materials include nonabsorbablematerials such as tantalum mesh, stainless steel mesh, polyester cloth,polyester sheeting, nylon mesh, Dacron mesh, acrylic cloth, polyvinylsponge, polytetrafluroethylene (PTFE), expanded PTFE, polyvinyl cloth,polypropylene mesh. Of these, polypropylene mesh, commercially availableas MARLEX® (Phillips Petroleum Company, Bartlesville, Okla.) or PROLENE®(Ehticon, Inc., Somerville, N.J.), is preferred. Exemplary absorbablemeshes include collagen, polyglycolic acid, polyglactin, and carbonfiber mesh.

The patch may be in the form of a single flat sheet or may be foldedinto a “plug” as is customarily used in tension free hernia repairmethods. Plug/patch combinations are also suitable for use with theadhesive composition. For example, the Prolene Hernia System™ (Ethicon,Somerville, N.J.), uses two sheets of polypropylene joined at a centerplug that are placed against the anterior and posterior surfaces of theherniated abdominal wall with the center plug passing through the areaof herniation. Other types of acceptable patches are well known to oneof skill in the art. U.S. Pat. No. 6,258,124 to Darois, U.S. Pat. No.5,147,374 to Fernadex, and U.S. Pat. No. 5,176,692 to Wilk et al.disclose several variations of hernia repair patches and methods.

The density, porosity, permeability, and thickness of the patch willvary for different patch types and different surgical applications. Ingeneral, a clean, dry, non-oily, rough surface is preferred. The patchneed not be functionalized as the patch may be entrapped in the adhesivecomposition matrix that is bound to the tissue surface. If desired, thepatch may however be functionalized with nucleophilic groups such as,but not limited to, amines, sulfhydryls, and the like. Such functionalgroups may serve to enhance the bonding strength of the adhesivecomposition. The patch may also be coated with the adhesive compositioncomponents in dry form. When such a coated patch is used, the adhesivecomposition begins to crosslink once exposed to moisture, such as bodyfluids, and so forth.

Administration and Use

The adhesive compositions of the present invention may be applied to anytissue surface and may be used in any customary method of tissue repair.The adhesive composition, as discussed below, is preferably appliedbefore crosslinking of the various components of the composition hasreached “equilibrium.” The point at which crosslinking has reachedequilibrium is defined herein as the point at which the composition nolonger feels tacky or sticky to the touch. The adhesive compositions ofthe present invention are generally delivered to the site ofadministration in such a way that the individual components of thecomposition come into contact with one another for the first time at thesite of administration, or within one hour preceding administration.

Thus, in one embodiment the compositions of the present invention aredelivered to the site of administration using an apparatus that allowsthe components to be delivered separately. Such delivery systems usuallyinvolve a multi-compartment spray device. Alternatively, the componentscan be delivered separately using any type of controllable extrusionsystem, or they can be delivered manually in the form of separatepastes, liquids or dry powders, and mixed together manually at the siteof administration. Many devices that are adapted for delivery ofmulti-component tissue sealants/hemostatic agents are well known in theart and can also be used in the practice of the present invention.

Yet another way of delivering the adhesive compositions of the presentinvention is to prepare the reactive components in inactive form aseither a liquid or powder. Such compositions can then be activated afterapplication to the tissue site, or immediately beforehand, by applyingan activator. In one embodiment, the activator is a buffer solutionhaving a pH that will activate the composition once mixed therewith.Still another way of delivering the compositions is to prepare preformedsheets, and apply the sheets as such to the site of administration. Oneof skill in the art can easily determine the appropriate administrationprotocol to use with any particular composition having a known gelstrength and gelation time. A more detailed description of the adhesivecomposition is given below.

The Adhesive Composition

The adhesive composition has a hydrophilic polymer component and aplurality of crosslinkable components. Additionally, other componentsmay also be present. A discussion of each of these components ispresented below.

The Hydrophilic Polymer Component

The hydrophilic polymer component may be a synthetic or naturallyoccurring hydrophilic polymer. Naturally occurring hydrophilic polymersinclude, but are not limited to: proteins such as collagen, fibronectin,albumins, globulins, fibrinogen, and fibrin, with collagen particularlypreferred; carboxylated polysaccharides such as polymannuronic acid andpolygalacturonic acid; aminated polysaccharides, particularly theglycosaminoglycans, e.g., hyaluronic acid, chitin, chondroitin sulfateA, B, or C, keratin sulfate, keratosulfate and heparin; and activatedpolysaccharides such as dextran and starch derivatives. Collagen andglycosaminoglycans are preferred naturally occurring hydrophilicpolymers for use herein.

In general, collagen from any source may be used in the adhesivecomposition of the method; for example, collagen may be extracted andpurified from human or other mammalian source, such as bovine or porcinecorium and human placenta, or may be recombinantly or otherwiseproduced. The preparation of purified, substantially non-antigeniccollagen in solution from bovine skin is well known in the art. Commonlyowned U.S. Pat. No. 5,428,022, to Palefsky et al., discloses methods ofextracting and purifying collagen from the human placenta. Commonlyowned U.S. Pat. No. 5,667,839, to Berg, discloses methods of producingrecombinant human collagen in the milk of transgenic animals, includingtransgenic cows. The term “collagen” or “collagen material” as usedherein refers to all forms of collagen, including those that have beenprocessed or otherwise modified.

Collagen of any type, including, but not limited to, types I, II, III,IV, or any combination thereof, may be used in the compositions of theinvention, although type I is generally preferred. Either atelopeptideor telopeptide-containing collagen may be used; however, when collagenfrom a source, such as bovine collagen, is used, atelopeptide collagenis generally preferred, because of its reduced immunogenicity comparedto telopeptide-containing collagen.

Collagen that has not been previously crosslinked by methods such asheat, irradiation, or chemical crosslinking agents is preferred for usein the compositions of the invention, although previously crosslinkedcollagen may be used. Non-crosslinked atelopeptide fibrillar collagen iscommercially available from McGhan Medical Corporation (Santa Barbara,Calif.) at collagen concentrations of 35 mg/ml and 65 mg/ml under thetrademarks ZYDERM® I Collagen and ZYDERM® II Collagen, respectively.Glutaraldehyde-crosslinked atelopeptide fibrillar collagen iscommercially available from McGhan Medical Corporation at a collagenconcentration of 35 mg/ml under the trademark ZYPLAST®.

Collagens for use in the present invention are generally, although notnecessarily, in aqueous suspension at a concentration between about 20mg/ml to about 120 mg/ml, preferably between about 30 mg/ml to about 90mg/ml.

Although intact collagen is preferred, denatured collagen, commonlyknown as gelatin, can also be used in the compositions of the invention.Gelatin may have the added benefit of being degradable faster thancollagen.

Because of its greater surface area and greater concentration ofreactive groups, nonfibrillar collagen is generally preferred. The term“nonfibrillar collagen” refers to any modified or unmodified collagenmaterial that is in substantially nonfibrillar form at pH 7, asindicated by optical clarity of an aqueous suspension of the collagen.

Collagen that is already in nonfibrillar form may be used in thecompositions of the invention. As used herein, the term “nonfibrillarcollagen” is intended to encompass collagen types that are nonfibrillarin native form, as well as collagens that have been chemically modifiedsuch that they are in nonfibrillar form at or around neutral pH.Collagen types that are nonfibrillar (or microfibrillar) in native forminclude types IV, VI, and VII.

Chemically modified collagens that are in nonfibrillar form at neutralpH include succinylated collagen, propylated collagen, ethylatedcollagen, methylated collagen, and the like, both of which can beprepared according to the methods described in U.S. Pat. No. 4,164,559,to Miyata et al., which is hereby incorporated by reference in itsentirety. Due to its inherent tackiness, methylated collagen isparticularly preferred, as disclosed in commonly owned U.S. Pat. No.5,614,587 to Rhee et al.

Collagens for use in the crosslinkable compositions of the presentinvention may start out in fibrillar form, then rendered nonfibrillar bythe addition of one or more fiber disassembly agents. The fiberdisassembly agent must be present in an amount sufficient to render thecollagen substantially nonfibrillar at pH 7, as described above. Fiberdisassembly agents for use in the present invention include, withoutlimitation, various biocompatible alcohols, amino acids, inorganicsalts, and carbohydrates, with biocompatible alcohols being particularlypreferred. Preferred biocompatible alcohols include glycerol andpropylene glycol. Non-biocompatible alcohols, such as ethanol, methanol,and isopropanol, are not preferred for use in the present invention, dueto their potentially deleterious effects on the body of the patientreceiving them. Preferred amino acids include arginine. Preferredinorganic salts include sodium chloride and potassium chloride. Althoughcarbohydrates, such as various sugars including sucrose, may be used inthe practice of the present invention, they are not as preferred asother types of fiber disassembly agents because they can have cytotoxiceffects in vivo.

As fibrillar collagen has less surface area and a lower concentration ofreactive groups than nonfibrillar, fibrillar collagen is less preferred.However, as disclosed in commonly owned, U.S. application Ser. No.08/476,825, now U.S. Pat. No. 5,614,587, fibrillar collagen, or mixturesof nonfibrillar and fibrillar collagen, may be preferred for use inadhesive compositions intended for long-term persistence in vivo, ifoptical clarity is not a requirement.

Synthetic hydrophilic polymers may also be used in the presentinvention. Useful synthetic hydrophilic polymers include, but are notlimited to: polyalkylene oxides, particularly polyethylene glycol andpoly(ethylene oxide)-poly(propylene oxide) copolymers, including blockand random copolymers; polyols such as glycerol, polyglycerol(particularly highly branched polyglycerol), propylene glycol andtrimethylene glycol substituted with one or more polyalkylene oxides,e.g., mono-, di- and tri-polyoxyethylated glycerol, mono- anddi-polyoxyethylated propylene glycol, and mono- and di-polyoxyethylatedtrimethylene glycol; polyoxyethylated sorbitol, polyoxyethylatedglucose; acrylic acid polymers and analogs and copolymers thereof, suchas polyacrylic acid per se, polymethacrylic acid,poly(hydroxyethyl-methacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxideacrylate) and copolymers of any of the foregoing, and/or with additionalacrylate species such as aminoethyl acrylate and mono-2-(acryloxy)-ethylsuccinate; polymaleic acid; poly(acrylamides) such as polyacrylamide perse, poly(methacrylamide), poly(dimethylacrylamide), andpoly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as poly(vinylalcohol); poly(N-vinyl lactams) such as poly(vinyl pyrrolidone),poly(N-vinyl caprolactam), and copolymers thereof; polyoxazolines,including poly(methyloxazoline) and poly(ethyloxazoline); andpolyvinylamines. It must be emphasized that the aforementioned list ofpolymers is not exhaustive, and a variety of other synthetic hydrophilicpolymers may be used, as will be appreciated by those skilled in theart.

The Crosslinkable Components

The adhesive composition also comprises a plurality of crosslinkablecomponents. Each of the crosslinkable components participates in areaction that results in a crosslinked matrix. Prior to completion ofthe crosslinking reaction, the crosslinkable components provide thenecessary adhesive qualities that enable the method of the invention.

The crosslinkable components are selected so that crosslinking givesrise to a biocompatible, nonimmunogenic matrix useful in a variety ofcontexts other than the presently claimed method, including adhesionprevention, biologically active agent delivery, tissue augmentation, andother applications. The crosslinkable components of the inventioncomprise: a component A, which has m nucleophilic groups, wherein m≧2and a component B, which has n electrophilic groups capable of reactionwith the m nucleophilic groups, wherein n≧2 and m+n≧4. An optional thirdcomponent, optional component C, which has at least one functional groupthat is either electrophilic and capable of reaction with thenucleophilic groups of component A, or nucleophilic and capable ofreaction with the electrophilic groups of component B may also bepresent. Thus, the total number of functional groups present oncomponents A, B and C, when present, in combination is greater than orequal to 5; that is, the total functional groups given by m+n+p must begreater than or equal to 5, where p is the number of functional groupson component C and, as indicated, is greater than or equal to 1. Each ofthe components is biocompatible and nonimmunogenic, and at least onecomponent is comprised of a hydrophilic polymer. Also, as will beappreciated, the adhesive composition may contain additionalcrosslinkable components D, E, F, etc., having one or more reactivenucleophilic or electrophilic groups and thereby participate information of the crosslinked biomaterial via covalent bonding to othercomponents.

The m nucleophilic groups on component A may all be the same, or,alternatively, A may contain two or more different nucleophilic groups.Similarly, the n electrophilic groups on component B may all be thesame, or two or more different electrophilic groups may be present. Thefunctional group(s) on optional component C, if nucleophilic, may or maynot be the same as the nucleophilic groups on component A, and,conversely, if electrophilic, the functional group(s) on optionalcomponent C may or may not be the same as the electrophilic groups oncomponent B.

Accordingly, the components may be represented by the structuralformulaeR¹(-[Q¹]_(q)-X)_(m) (component A),  (I)R²(-[Q²]_(r)-Y)_(n) (component B), and  (II)R³(-[Q³]_(s)-Fn)_(p) (optional component C),  (III)wherein:

-   -   R¹, R² and R³ are independently selected from the group        consisting of C₂ to C₁₄ hydrocarbyl, heteroatom-containing C₂ to        C₁₄ hydrocarbyl, hydrophilic polymers, and hydrophobic polymers,        providing that at least one of R¹, R² and R³ is a hydrophilic        polymer, preferably a synthetic hydrophilic polymer;    -   X represents one of the m nucleophilic groups of component A,        and the various X moieties on A may be the same or different;    -   Y represents one of the n electrophilic groups of component B,        and the various Y moieties on A may be the same or different;    -   Fn represents a functional group on optional component C;    -   Q¹, Q² and Q³ are linking groups;    -   m≧2, n≧2, m+n is ≧4, q, and r are independently zero or 1, and        when optional component C is present, p≧1, and s is        independently zero or 1.

Reactive Groups

X may be virtually any nucleophilic group, so long as reaction can occurwith the electrophilic group Y. Analogously, Y may be virtually anyelectrophilic group, so long as reaction can take place with X. The onlylimitation is a practical one, in that reaction between X and Y shouldbe fairly rapid and take place automatically upon admixture with anaqueous medium, without need for heat or potentially toxic ornon-biodegradable reaction catalysts or other chemical reagents. It isalso preferred although not essential that reaction occur without needfor ultraviolet or other radiation. Ideally, the reactions between X andY should be complete in under 60 minutes, preferably under 30 minutes.Most preferably, the reaction occurs in about 5 to 15 minutes or less.

Examples of nucleophilic groups suitable as X include, but are notlimited to, —NH₂, —NHR⁴, —N(R⁴)₂, —SH, —OH, —COOH, —C₆, H—OH, —PH₂,—PHR⁵, —P(R⁵)₂, —NH—NH₂, —CO—NH—NH₂, —C₅H₄N, etc. wherein R⁴ and R⁵ arehydrocarbyl, typically alkyl or monocyclic aryl, preferably alkyl, andmost preferably lower alkyl. Organometallic moieties are also usefulnucleophilic groups for the purposes of the invention, particularlythose that act as carbanion donors. Organometallic nucleophiles are not,however, preferred. Examples of organometallic moieties include:Grignard functionalities —R⁶MgHal wherein R⁶ is a carbon atom(substituted or unsubstituted), and Hal is halo, typically bromo, iodoor chloro, preferably bromo; and lithium-containing functionalities,typically alkyllithium groups; sodium-containing functionalities.

It will be appreciated by those of ordinary skill in the art thatcertain nucleophilic groups must be activated with a base so as to becapable of reaction with an electrophile. For example, when there arenucleophilic sulfhydryl and hydroxyl groups in the crosslinkablecomposition, the composition must be admixed with an aqueous base inorder to remove a proton and provide an —S⁻ or —O⁻ species to enablereaction with an electrophile. Unless it is desirable for the base toparticipate in the crosslinking reaction, a normucleophilic base ispreferred. In some embodiments, the base may be present as a componentof a buffer solution. Suitable bases and corresponding crosslinkingreactions are described infra in Section E.

The selection of electrophilic groups provided within the crosslinkablecomposition, i.e., on component B, must be made so that reaction ispossible with the specific nucleophilic groups. Thus, when the Xmoieties are amino groups, the Y groups are selected so as to react withamino groups. Analogously, when the X moieties are sulfhydryl moieties,the corresponding electrophilic groups are sulfhydryl-reactive groups,and the like.

By way of example, when X is amino (generally although not necessarilyprimary amino), the electrophilic groups present on Y are amino reactivegroups such as, but not limited to: (1) carboxylic acid esters,including cyclic esters and “activated” esters; (2) acid chloride groups(—CO—Cl); (3) anhydrides (—(CO)—O—(CO)—R); (4) ketones and aldehydes,including α,β-unsaturated aldehydes and ketones such as —CH═CH—CH═O and—CH═CH—C(CH₃)═O; (5) halides; (6) isocyanate (—N═C═O); (7)isothiocyanate (—N═C=S); (8) epoxides; (9) activated hydroxyl groups(e.g., activated with conventional activating agents such ascarbonyldiimidazole or sulfonyl chloride); and (10) olefins, includingconjugate olefins, such as ethenesulfonyl (—SO₂CH═CH₂) and analogousfunctional groups, including acrylate (—CO₂—C═CH₂), methacrylate(—CO₂—C(CH₃)═CH₂)), ethyl acrylate (—CO₂—C(CH₂CH₃)═CH₂), andethyleneimino (—CH═CH—C═NH). Since a carboxylic acid group per se is notsusceptible to reaction with a nucleophilic amine, components containingcarboxylic acid groups must be activated so as to be amine-reactive.Activation may be accomplished in a variety of ways, but often involvesreaction with a suitable hydroxyl-containing compound in the presence ofa dehydrating agent such as dicyclohexylcarbodiimide (DCC) ordicyclohexylurea (DHU). For example, a carboxylic acid can be reactedwith an alkoxy-substituted N-hydroxy-succinimide orN-hydroxysulfosuccinimide in the presence of DCC to form reactiveelectrophilic groups, the N-hydroxysuccinimide ester and theN-hydroxysulfosuccinimide ester, respectively. Carboxylic acids may alsobe activated by reaction with an acyl halide such as an acyl chloride(e.g., acetyl chloride), to provide a reactive anhydride group. In afurther example, a carboxylic acid may be converted to an acid chloridegroup using, e.g., thionyl chloride or an acyl chloride capable of anexchange reaction. Specific reagents and procedures used to carry outsuch activation reactions will be known to those of ordinary skill inthe art and are described in the pertinent texts and literature.

Analogously, when X is sulfhydryl, the electrophilic groups present on Yare groups that react with a sulfhydryl moiety. Such reactive groupsinclude those that form thioester linkages upon reaction with asulflhydryl group, such as those described in applicants' PCTPublication No. WO 00/62827 to Wallace et al. As explained in detailtherein, such “sulfhydryl reactive” groups include, but are not limitedto: mixed anhydrides; ester derivatives of phosphorus; ester derivativesof p-nitrophenol, p-nitrothiophenol and pentafluorophenol; esters ofsubstituted hydroxylamines, including N-hydroxyphthalimide esters,N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, andN-hydroxyglutarimide esters; esters of 1-hydroxybenzotriazole;3-hydroxy-3,4-dihydro-benzotriazin-4-one;3-hydroxy-3,4-dihydro-quinazoline-4-one; carbonylimidazole derivatives;acid chlorides; ketenes; and isocyanates. With these sulfhydryl reactivegroups, auxiliary reagents can also be used to facilitate bondformation, e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide can beused to facilitate coupling of sulfhydryl groups to carboxyl-containinggroups.

In addition to the sulfhydryl reactive groups that form thioesterlinkages, various other sulfhydryl reactive functionalities can beutilized that form other types of linkages. For example, compounds thatcontain methyl imidate derivatives form imido-thioester linkages withsulfhydryl groups. Alternatively, sulfhydryl reactive groups can beemployed that form disulfide bonds with sulfhydryl groups; such groupsgenerally have the structure —S—S—Ar where Ar is a substituted orunsubstituted nitrogen-containing heteroaromatic moiety or anon-heterocyclic aromatic group substituted with an electron-withdrawingmoiety, such that Ar may be, for example, 4-pyridinyl, o-nitrophenyl,m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2-nitro-4-benzoic acid,2-nitro-4-pyridinyl, etc. In such instances, auxiliary reagents, i.e.,mild oxidizing agents such as hydrogen peroxide, can be used tofacilitate disulfide bond formation.

Yet another class of sulfhydryl reactive groups forms thioether bondswith sulfhydryl groups. Such groups include, inter alia, maleimido,substituted maleimido, haloalkyl, epoxy, imino, and aziridino, as wellas olefins (including conjugated olefins) such as ethenesulfonyl,etheneimino, acrylate, methacrylate, and α,β-unsaturated aldehydes andketones. This class of sulfhydryl reactive groups are particularlypreferred as the thioether bonds may provide faster crosslinking andlonger in vivo stability.

When X is —OH, the electrophilic functional groups on the remainingcomponent(s) must react with hydroxyl groups. The hydroxyl group may beactivated as described above with respect to carboxylic acid groups, orit may react directly in the presence of base with a sufficientlyreactive electrophile such as an epoxide group, an aziridine group, anacyl halide, or an anhydride.

When X is an organometallic nucleophile such as a Grignard functionalityor an alkyllithium group, suitable electrophilic functional groups forreaction therewith are those containing carbonyl groups, including, byway of example, ketones and aldehydes.

It will also be appreciated that certain functional groups can react asnucleophiles or as electrophiles, depending on the selected reactionpartner and/or the reaction conditions. For example, a carboxylic acidgroup can act as a nucleophile in the presence of a fairly strong base,but generally acts as an electrophile allowing nucleophilic attack atthe carbonyl carbon and concomitant replacement of the hydroxyl groupwith the incoming nucleophile.

The covalent linkages in the crosslinked structure that result uponcovalent binding of specific nucleophilic components to specificelectrophilic components in the crosslinkable composition include,solely by way of example, the following (the optional linking groups Q¹and Q² are omitted for clarity):

TABLE 1 REPRESENTATIVE NUCLEOPHILIC COMPONENT (A, optionalREPRESENTATIVE component C ELECTROPHILIC COMPONENT element FN_(NU)) (B,FN_(EL)) RESULTING LINKAGE R¹—NH₂ R²—O—(CO)—O—N(COCH₂) R¹—NH—(CO)—O—R²(succinimidyl carbonate terminus) R¹—SH R²—O—(CO)—O—N(COCH₂)R¹—S—(CO)—O—R² R¹—OH R²—O—(CO)—O—N(COCH₂) R¹—O—(CO)—O—R² R¹—NH₂R²—O(CO)—CH═CH₂ R¹—NH—CH₂CH₂—(CO)—O—R² (acrylate terminus) R¹—SHR²—O(CO)—CH═CH₂ R¹—S—CH₂CH₂—(CO)—O—R² R¹—OH R²—O(CO)—CH═CH₂R¹—O—CH₂CH₂—(CO)—O—R² R¹—NH₂ R²—O(CO)—(CH₂)₃—CO₂—N(COCH₂)R¹—NH—(CO)—(CH₂)₃—(CO)—OR² (succinimidyl glutarate terminus) R¹—SHR²—O(CO)—(CH₂)₃—CO₂—N(COCH₂) R¹—S—(CO)—(CH₂)₃—(CO)—OR² R¹—OHR²—O(CO)—(CH₂)₃—CO₂—N(COCH₂) R¹—O—(CO)—(CH₂)₃—(CO)—OR² R¹—NH₂R²—O—CH₂—CO₂—N(COCH₂) R¹—NH—(CO)—CH₂—OR² (succinimidyl acetate terminus)R¹—SH R²—O—CH₂—CO₂—N(COCH₂) R¹—S—(CO)—CH₂—OR² R¹—OHR²—O—CH₂—CO₂—N(COCH₂) R¹—O—(CO)—CH₂—OR² R¹—NH₂ R²—O—NH(CO)—(CH₂)₂—CO₂—R¹—NH—(CO)—(CH₂)₂—(CO)—NH—OR² N(COCH₂) (succinimidyl succinamideterminus) R¹—SH R²—O—NH(CO)—(CH₂)₂—CO₂— R¹—S—(CO)—(CH₂)₂—(CO)—NH—OR²N(COCH₂) R¹—OH R²—O—NH(CO)—(CH₂)₂—CO₂— R¹—O—(CO)—(CH₂)₂—(CO)—NH—OR²N(COCH₂) R¹—NH₂ R²—O—(CH₂)₂—CHO R¹—NH—(CO)—(CH₂)₂—OR² (propionaldehydeterminus) R¹—NH₂

R¹—NH—CH₂—CH(OH)—CH₂—OR² and R¹—N[CH₂—CH(OH)—CH₂—OR²]₂ (glycidyl etherterminus) R¹—NH₂ R²—SO₂—CH═CH₂ R¹—NH—CH₂CH₂—SO₂—R² (vinyl sulfoneterminus) R¹—SH R²—SO₂—CH═CH₂ R¹—S—CH₂CH₂—SO₂—R²

Linking Groups

The functional groups X and Y and FN on optional component C may bedirectly attached to the compound core (R¹, R² or R³ on optionalcomponent C, respectively), or they may be indirectly attached through alinking group, with longer linking groups also termed “chain extenders.”In structural formulae (I), (II) and (III), the optional linking groupsare represented by Q¹, Q² and Q³, wherein the linking groups are presentwhen q, r and s are equal to 1 (with R, X, Y, Fn, m n and p as definedpreviously).

Suitable linking groups are well known in the art. See, for example,International Pat. Publication No. WO 97/22371. Linking groups areuseful to avoid steric hindrance problems that are sometimes associatedwith the formation of direct linkages between molecules. Linking groupsmay additionally be used to link several multifunctionally activatedcompounds together to make larger molecules. In a preferred embodiment,a linking group can be used to alter the degradative properties of thecompositions after administration and resultant gel formation. Forexample, linking groups can be incorporated into components A, B, oroptional component C to promote hydrolysis, to discourage hydrolysis, orto provide a site for enzymatic degradation.

Examples of linking groups that provide hydrolyzable sites, include,inter alia: ester linkages; anhydride linkages, such as obtained byincorporation of glutarate and succinate; ortho ester linkages; orthocarbonate linkages such as trimethylene carbonate; amide linkages;phosphoester linkages; α-hydroxy acid linkages, such as may be obtainedby incorporation of lactic acid and glycolic acid; lactone-basedlinkages, such as may be obtained by incorporation of caprolactone,valerolactone, y-butyrolactone and p-dioxanone; and amide linkages suchas in a dimeric, oligomeric, or poly(amino acid) segment. Examples ofnon-degradable linking groups include succinimide, propionic acid andcarboxymethylate linkages. See, for example, PCT WO 99/07417. Examplesof enzymatically degradable linkages include Leu-Gly-Pro-Ala, which isdegraded by collagenase; and Gly-Pro-Lys, which is degraded by plasmin.

Linking groups can also enhance or suppress the reactivity of thevarious nucleophilic and electrophilic groups. For example,electron-withdrawing groups within one or two carbons of a sulfhydrylgroup would be expected to diminish its effectiveness in coupling, dueto a lowering of nucleophilicity. Carbon-carbon double bonds andcarbonyl groups will also have such an effect. Conversely,electron-withdrawing groups adjacent to a carbonyl group (e.g., thereactive carbonyl of glutaryl-N-hydroxysuccinimidyl) would increase thereactivity of the carbonyl carbon with respect to an incomingnucleophile. By contrast, sterically bulky groups in the vicinity of afunctional group can be used to diminish reactivity and thus couplingrate as a result of steric hindrance.

By way of example, particular linking groups and corresponding componentstructure are indicated in Table 2:

TABLE 2 LINKING GROUP COMPONENT STRUCTURE —O—(CH₂)_(n)— Component A:R¹—O—(CH₂)_(n)—X Component B: R²—O—(CH₂)_(n)—Y Optional Component C:R³—O—(CH₂)_(n)—Z —S—(CH₂)_(n)— Component A: R¹—S—(CH₂)_(n)—X ComponentB: R²—S—(CH₂)_(n)—Y Optional Component C: R³—S—(CH₂)_(n)—Z—NH—(CH₂)_(n)— Component A: R¹—NH—(CH₂)_(n)—X Component B:R²—NH—(CH₂)_(n)—Y Optional Component C: R³—NH—(CH₂)_(n)—Z—O—(CO)—NH—(CH₂)_(n)— Component A: R¹—O—(CO)—NH—(CH₂)_(n)—X Component B:R²—O—(CO)—NH—(CH₂)_(n)—Y Optional Component C: R³—O—(CO)—NH—(CH₂)_(n)—Z—NH—(CO)—O—(CH₂)_(n)— Component A: R¹—NH—(CO)—O—(CH₂)_(n)—X Component B:R²—NH—(CO)—O—(CH₂)_(n)—Y Optional Component C: R³—NH—(CO)—O—(CH₂)_(n)—Z—O—(CO)—(CH₂)_(n)— Component A: R¹—O—(CO)—(CH₂)_(n)—X Component B:R²—O—(CO)—(CH₂)_(n)—Y Optional Component C: R³—O—(CO)—(CH₂)_(n)—Z—(CO)—O—(CH₂)_(n)— Component A: R¹—(CO)—O—(CH₂)_(n)—X Component B:R²—(CO)—O—(CH₂)_(n)—Y Optional Component C: R³—(CO)—O—(CH₂)_(n)—Z—O—(CO)—O—(CH₂)_(n)— Component A: R¹—O—(CO)—O—(CH₂)_(n)—X Component B:R²—O—(CO)—O—(CH₂)_(n)—Y Optional Component C: R³—O—(CO)—O—(CH₂)_(n)—Z—O—(CO)—CHR⁷— Component A: R¹—O—(CO)—CHR⁷—X Component B:R²—O—(CO)—CHR⁷—Y Optional Component C: R³—O—(CO)—CHR⁷—Z —O—R⁸—(CO)—NH—Component A: R¹—O—R⁸—(CO)—NH—X Component B: R²—O—R⁸—(CO)—NH—Y OptionalComponent C: R³—O—R⁸—(CO)—NH—Z

In the table, n is generally in the range of 1 to about 10, R⁷ isgenerally hydrocarbyl, typically alkyl or aryl, preferably alkyl, andmost preferably lower alkyl, and R⁸ is hydrocarbylene,heteroatom-containing hydrocarbylene, substituted hydrocarbylene, orsubstituted heteroatom-containing hydrocarbylene) typically alkylene orarylene (again, optionally substituted and/or containing a heteroatom),preferably lower alkylene (e.g., methylene, ethylene, n-propylene,n-butylene, etc.), phenylene, or amidoalkylene (e.g., —(CO)—NH—CH₂).

Other general principles that should be considered with respect tolinking groups are as follows: If higher molecular weight components areto be used, they preferably have biodegradable linkages as describedabove, so that fragments larger than 20,000 mol. wt. are not generatedduring resorption in the body. In addition, to promote water miscibilityand/or solubility, it may be desired to add sufficient electric chargeor hydrophilicity. Hydrophilic groups can be easily introduced usingknown chemical synthesis, so long as they do not give rise to unwantedswelling or an undesirable decrease in compressive strength. Inparticular, polyalkoxy segments may weaken gel strength.

The Component Core

The “core” of each crosslinkable component is comprised of the molecularstructure to which the nucleophilic or electrophilic groups are bound.Using the formulae (I) R¹-[Q¹]_(q)—X)_(m), for component A, (II)R²(-[Q²]_(r)—Y)_(n) for component B, and (III) R³(-[Q³]_(s)—Fn)_(p) foroptional component C, the “core” groups are R¹, R² and R³. Eachmolecular core of the reactive components of the crosslinkablecomposition is generally selected from synthetic and naturally occurringhydrophilic polymers, hydrophobic polymers, and C₂-C₁₄ hydrocarbylgroups zero to 2 heteroatoms selected from N, O and S, with the provisothat at least one of the crosslinkable components A, B, and optionallyC, comprises a molecular core of a synthetic hydrophilic polymer. In apreferred embodiment, at least one of A and B comprises a molecular coreof a synthetic hydrophilic polymer.

Hydrophilic Polymers and “Activation” Thereof

A “hydrophilic polymer” as used herein refers to a synthetic polymerhaving an average molecular weight and composition effective to renderthe polymer “hydrophilic” as defined in Part (I) of this section. Asdiscussed above, synthetic hydrophilic polymers useful herein include,but are not limited to: polyalkylene oxides, particularly polyethyleneglycol and poly(ethylene oxide)-poly(propylene oxide) copolymers,including block and random copolymers; polyols such as glycerol,polyglycerol (particularly highly branched polyglycerol), propyleneglycol and trimethylene glycol substituted with one or more polyalkyleneoxides, e.g., mono-, di- and tri-polyoxyethylated glycerol, mono- anddi-polyoxyethylated propylene glycol, and mono- and di-polyoxyethylatedtrimethylene glycol; polyoxyethylated sorbitol, polyoxyethylatedglucose; acrylic acid polymers and analogs and copolymers thereof, suchas polyacrylic acid per se, polymethacrylic acid,poly(hydroxyethyl-methacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxideacrylate) and copolymers of any of the foregoing, and/or with additionalacrylate species such as aminoethyl acrylate and mono-2-(acryloxy)-ethylsuccinate; polymaleic acid; poly(acrylamides) such as polyacrylamide perse, poly(methacrylamide), poly(dimethylacrylamide), andpoly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as poly(vinylalcohol); poly(N-vinyl lactams) such as poly(vinyl pyrrolidone),poly(N-vinyl caprolactam), and copolymers thereof; polyoxazolines,including poly(methyloxazoline) and poly(ethyloxazoline); andpolyvinylamines. It must be emphasized that the aforementioned list ofpolymers is not exhaustive, and a variety of other synthetic hydrophilicpolymers may be used, as will be appreciated by those skilled in theart.

The synthetic hydrophilic polymer may be a homopolymer, a blockcopolymer, a random copolymer, or a graft copolymer. In addition, thepolymer may be linear or branched, and if branched, may be minimally tohighly branched, dendrimeric, hyperbranched, or a star polymer. Thepolymer may include biodegradable segments and blocks, eitherdistributed throughout the polymer's molecular structure or present as asingle block, as in a block copolymer. Biodegradable segments are thosethat degrade so as to break covalent bonds. Typically, biodegradablesegments are segments that are hydrolyzed in the presence of waterand/or enzymatically cleaved in situ. Biodegradable segments may becomposed of small molecular segments such as ester linkages, anhydridelinkages, ortho ester linkages, ortho carbonate linkages, amidelinkages, phosphonate linkages, etc. Larger biodegradable “blocks” willgenerally be composed of oligomeric or polymeric segments incorporatedwithin the hydrophilic polymer. Illustrative oligomeric and polymericsegments that are biodegradable include, by way of example, poly(aminoacid) segments, poly(orthoester) segments, poly(orthocarbonate)segments, and the like.

Other suitable synthetic hydrophilic polymers include chemicallysynthesized polypeptides, particularly polynucleophilic polypeptidesthat have been synthesized to incorporate amino acids containing primaryamino groups (such as lysine) and/or amino acids containing thiol groups(such as cysteine). Poly(lysine), a synthetically produced polymer ofthe amino acid lysine (145 MW), is particularly preferred. Poly(lysine)shave been prepared having anywhere from 6 to about 4,000 primary aminogroups, corresponding to molecular weights of about 870 to about580,000. Poly(lysine)s for use in the present invention preferably havea molecular weight within the range of about 1,000 to about 300,000,more preferably within the range of about 5,000 to about 100,000, andmost preferably, within the range of about 8,000 to about 15,000.Poly(lysine)s of varying molecular weights are commercially availablefrom Peninsula Laboratories, Inc. (Belmont, Calif.).

The synthetic hydrophilic polymer may be a homopolymer, a blockcopolymer, a random copolymer, or a graft copolymer. In addition, thepolymer may be linear or branched, and if branched, may be minimally tohighly branched, dendrimeric, hyperbranched, or a star polymer. Thepolymer may include biodegradable segments and blocks, eitherdistributed throughout the polymer's molecular structure or present as asingle block, as in a block copolymer. Biodegradable segments are thosethat degrade so as to break covalent bonds. Typically, biodegradablesegments are segments that are hydrolyzed in the presence of waterand/or enzymatically cleaved in situ. Biodegradable segments may becomposed of small molecular segments such as ester linkages, anhydridelinkages, ortho ester linkages, ortho carbonate linkages, amidelinkages, phosphonate linkages, etc. Larger biodegradable “blocks” willgenerally be composed of oligomeric or polymeric segments incorporatedwithin the hydrophilic polymer. Illustrative oligomeric and polymericsegments that are biodegradable include, by way of example, poly(aminoacid) segments, poly(orthoester) segments, poly(orthocarbonate)segments, and the like.

Although a variety of different synthetic hydrophilic polymers can beused in the present compositions, as indicated above, preferredsynthetic hydrophilic polymers are polyethylene glycol (PEG) andpolyglycerol (PG), particularly highly branched polyglycerol. Variousforms of PEG are extensively used in the modification of biologicallyactive molecules because PEG lacks toxicity, antigenicity, andimmunogenicity (i.e., is biocompatible), can be formulated so as to havea wide range of solubilities, and does not typically interfere with theenzymatic activities and/or conformations of peptides. A particularlypreferred synthetic hydrophilic polymer for certain applications is apolyethylene glycol (PEG) having a molecular weight within the range ofabout 100 to about 100,000 mol. wt., although for highly branched PEG,far higher molecular weight polymers can be employed—up to 1,000,000 ormore—providing that biodegradable sites are incorporated ensuring thatall degradation products will have a molecular weight of less than about30,000. For most PEGs, however, the preferred molecular weight is about1,000 to about 20,000 mol. wt., more preferably within the range ofabout 7,500 to about 20,000 mol. wt. Most preferably, the polyethyleneglycol has a molecular weight of approximately 10,000 mol. wt.

Naturally occurring hydrophilic polymers include, but are not limitedto: proteins such as collagen, fibronectin, albumins, globulins,fibrinogen, and fibrin, with collagen particularly preferred;carboxylated polysaccharides such as polymannuronic acid andpolygalacturonic acid; aminated polysaccharides, particularly theglycosaminoglycans, e.g., hyaluronic acid, chitin, chondroitin sulfateA, B, or C, keratin sulfate, keratosulfate and heparin; and activatedpolysaccharides such as dextran and starch derivatives. Collagen andglycosaminoglycans are preferred naturally occurring hydrophilicpolymers for use herein.

Any of the hydrophilic polymers herein must contain, or be activated tocontain, functional groups, i.e., nucleophilic or electrophilic groups,which enable crosslinking. Activation of PEG is discussed below; it isto be understood, however, that the following discussion is for purposesof illustration and analogous techniques may be employed with otherpolymers.

With respect to PEG, first of all, various functionalized polyethyleneglycols have been used effectively in fields such as proteinmodification (see Abuchowski et al., Enzymes as Drugs, John Wiley &Sons: New York, N.Y. (1981) pp. 367-383; and Dreborg et al., Crit. Rev.Therap. Drug Carrier Syst. (1990) 6:315), peptide chemistry (see Mutteret al., The Peptides, Academic: New York, N.Y. 2:285-332; and Zalipskyet al., Int. J. Peptide Protein Res. (1987) 30:740), and the synthesisof polymeric drugs (see Zalipsky et al., Eur. Polym. J. (1983) 19:1177;and Ouchi et al., J. Macromol. Sci. Chem. (1987) A24: 1011).

Activated forms of PEG, including multifunctionally activated PEG, arecommercially available, and are also easily prepared using knownmethods. For example, see Chapter 22 of Poly(ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, J. Milton Harris, ed., PlenumPress, NY (1992); and Shearwater Polymers, Inc. Catalog, PolyethyleneGlycol Derivatives, Huntsville, Ala. (1997-1998).

Structures for some specific, tetrafunctionally activated forms of PEGare shown in FIGS. 1 to 10, as are generalized reaction productsobtained by reacting the activated PEGs with multi-amino PEGs, i.e., aPEG with two or more primary amino groups. The activated PEGsillustrated have a pentaerythritol(2,2-bis(hydroxymethyl)-1,3-propanediol) core. Such activated PEGs, aswill be appreciated by those in the art, are readily prepared byconversion of the exposed hydroxyl groups in the PEGylated polyol (i.e.,the terminal hydroxyl groups on the PEG chains) to carboxylic acidgroups (typically by reaction with an anhydride in the presence of anitrogenous base), followed by esterification with N-hydroxysuccinimide,N-hydroxysulfosuccinimide, or the like, to give the polyfunctionallyactivated PEG.

FIG. 1 shows the reaction of tetrafunctionally activated PEGsuccinimidyl glutarate, referred to herein as “SG-PEG,” with multi-aminoPEG, and the reaction product obtained thereby.

Another activated form of PEG is PEG succinimidyl propionate (“SE-PEG”).The structural formula for tetrafunctionally activated SE-PEG and thereaction product obtained upon reaction with multi-amino PEG are shownin FIG. 2.

Analogous activated forms of PEG are PEG succinimidyl butylate and PEGsuccinimidyl acetate, the structures of which are shown in FIGS. 3 and4, respectively, along with the reaction products obtained upon reactionwith multi-amino PEG. SE-PEG, PEG succinimidyl butylate, and PEGsuccinimidyl acetate are sometimes referred to as “PEG succinimidyl”(PEG-S); see U.S. Pat. No. 5,328,955 to Rhee et al.

Another functionally activated form of PEG is referred to as “PEGsuccinimidyl succinamide” (SSA-PEG). The structural formula for thetetrafunctionally activated form of this compound and the reactionproduct obtained by reacting it with multi-amino PEG are shown in FIG.5. In the structure of FIG. 5, an ethylene (—CH₂CH₂—) group is shownadjacent to the succinimidyl ester; it is to be understood, however,that as with the PEG succinimidyl compounds, related structurescontaining a methylene linkage, an n-propylene linkage, or the like, arealso possible.

Yet another activated form of PEG is PEG succinimidyl carbonate(SC-PEG). The structural formula of tetrafunctionally activated SC-PEGand the conjugate formed by reacting it with multi-amino PEG are shownin FIG. 6.

PEG can also be derivatized to form functionally activated PEGpropionaldehyde (A-PEG), the tetrafunctionally activated form of whichis shown in FIG. 7, as is the conjugate formed by the reaction of A-PEGwith multi-amino PEG.

Yet another form of activated polyethylene glycol is functionallyactivated PEG glycidyl ether (E-PEG), of which the tetrafunctionallyactivated compound is shown in FIG. 8, as is the conjugate formed byreacting such with multi-amino PEG.

Another activated derivative of polyethylene glycol is functionallyactivated PEG-isocyanate (I-PEG), which is shown in FIG. 9, along withthe conjugate formed by reacting such with multi-amino PEG.

Another activated derivative of polyethylene glycol is functionallyactivated PEG-vinylsulfone (V-PEG), which is shown in FIG. 10, alongwith the conjugate formed by reacting such with multi-amino PEG.

Activation with succinimidyl groups to convert terminal carboxylic acidgroups to reactive esters is one technique for preparing a synthetichydrophilic polymer with electrophilic moieties suitable for reactionwith nucleophiles such as primary amines, thiols, and hydroxyl groups.Other activating agents for hydroxyl groups include carbonyldiimidazoleand sulfonyl chloride. However, as discussed in part (B) of thissection, a wide variety of electrophilic groups may be advantageouslyemployed for reaction with corresponding nucleophiles. Examples of suchelectrophilic groups include acid chloride groups; anhydrides, ketones,aldehydes, isocyanate, isothiocyanate, epoxides, and olefins, includingconjugated olefins such as ethenesulfonyl(—SO₂CH═CH₂) and analogousfunctional groups.

Hydrophilic di- or poly-nucleophilic polymers of the present compositionare exemplified in FIGS. 1-10 by multi-amino PEG. Various forms ofmulti-amino PEG are commercially available from Shearwater Polymers(Huntsville, Ala.) and from Texaco Chemical Company (Houston, Tex.)under the name “Jeffamine”. Multi-amino PEGs useful in the presentinvention include Texaco's Jeffamine diamines (“D” series) and triamines(“T” series), which contain two and three primary amino groups permolecule. Analogous poly(sulfhydryl) PEGs are also available fromShearwater Polymers, e.g., in the form of pentaerythritol poly(ethyleneglycol) ether tetra-sulfhydryl (molecular weight 10,000).

Hydrophobic Polymers

The crosslinkable compositions of the invention can also includehydrophobic polymers, although for most uses hydrophilic polymers arepreferred. Polylactic acid and polyglycolic acid are examples of twohydrophobic polymers that can be used. With other hydrophobic polymers,only short-chain oligomers should be used, containing at most about 14carbon atoms, to avoid solubility-related problems during reaction.

Low Molecular Weight Components

As indicated above, the molecular core of one or more of thecrosslinkable components can also be a low molecular weight compound,i.e., a C₂-C₁₄ hydrocarbyl group containing zero to 2 heteroatomsselected from N, O, S and combinations thereof. Such a molecular corecan be substituted with nucleophilic groups or with electrophilicgroups.

When the low molecular weight molecular core is substituted with primaryamino groups, the component may be, for example, ethylenediamine(H₂N—CH₂CH₂—NH₂), tetramethylenediamine (H₂N—(CH₄)—NH₂),pentamethylenediamine (cadaverine) (H₂N—(CH₅)—NH₂), hexamethylenediamine(H₂N—(CH₆)—NH₂), bis(2-aminoethyl)amine (HN-[CH₂CH₂—NH₂]₂), ortris(2-aminoethyl)amine (N-[CH₂CH₂—NH₂]₃).

Low molecular weight diols and polyols include trimethylolpropane,di(trimethylol propane), pentaerythritol, and diglycerol, all of whichrequire activation with a base in order to facilitate their reaction asnucleophiles. Such diols and polyols may also be functionalized toprovide di- and poly-carboxylic acids, functional groups that are, asnoted earlier herein, also useful as nucleophiles under certainconditions. Polyacids for use in the present compositions include,without limitation, trimethylolpropane-based tricarboxylic acid,di(trimethylol propane)-based tetracarboxylic acid, heptanedioic acid,octanedioic acid (suberic acid), and hexadecanedioic acid (thapsicacid), all of which are commercially available and/or readilysynthesized using known techniques.

Low molecular weight di- and poly-electrophiles include, for example,disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS₃),dithiobis(succinimidylpropionate) (DSP),bis(2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES), and3,3′-dithiobis(sulfosuccinimidylpropionate (DTSPP), and their analogsand derivatives. The aforementioned compounds are commercially availablefrom Pierce (Rockford, Ill.). Such di- and poly-electrophiles can alsobe synthesized from di- and polyacids, for example by reaction with anappropriate molar amount of N-hydroxysuccinimide in the presence of DCC.Polyols such as trimethylolpropane and di(trimethylol propane) can beconverted to carboxylic acid form using various known techniques, thenfurther derivatized by reaction with NHS in the presence of DCC toproduce trifunctionally and tetrafunctionally activated polymers.

Storage and Handling

Because crosslinkable components containing electrophilic groups reactwith water, the electrophilic component or components are generallystored and used in sterile, dry form to prevent hydrolysis. Processesfor preparing synthetic hydrophilic polymers containing multipleelectrophilic groups in sterile, dry form are set forth in commonlyassigned U.S. Pat. No. 5,643,464 to Rhee et al. For example, the drysynthetic polymer may be compression molded into a thin sheet ormembrane, which can then be sterilized using gamma or, preferably,e-beam irradiation. The resulting dry membrane or sheet can be cut tothe desired size or chopped into smaller size particulates.

Components containing multiple nucleophilic groups are generally notwater-reactive and can therefore be stored either dry or in aqueoussolution. If stored as a dry, particulate, solid, the various componentsof the crosslinkable composition may be blended and stored in a singlecontainer. Admixture of all components with water, saline, or otheraqueous media should not occur until immediately prior to use.

In an alternative embodiment, the crosslinking components can be mixedtogether in a single aqueous medium in which they are both unreactive,i.e. such as in a low pH buffer. Thereafter, they can be sprayed ontothe targeted tissue site along with a high pH buffer, after which theywill rapidly react and form a gel.

Suitable liquid media for storage of crosslinkable compositions includeaqueous buffer solutions such as monobasic sodium phosphate/dibasicsodium phosphate, sodium carbonate/sodium bicarbonate, glutamate oracetate, at a concentration of 0.5 to 300 mM. In general, asulfhydryl-reactive component such as PEG substituted with maleimidogroups or succinimidyl esters is prepared in water or a dilute buffer,with a pH of between around 5 to 6. Buffers with pKs between about 8 and10.5 for preparing a polysulfhydryl component such as sulfhydryl-PEG areuseful to achieve fast gelation time of compositions containing mixturesof sulfhydryl-PEG and SG-PEG. These include carbonate, borate and AMPSO(3-[(1,1-dimethyl-2-hydroxyethyl)amino]2-hydroxy-propane-sulfonic acid).In contrast, using a combination of maleimidyl PEG and sulfhydryl-PEG, apH of around 5 to 9 is preferred for the liquid medium used to preparethe sulfhydryl PEG.

Other Components

In order to enhance adhesive strength, it may be generally desirable toadd a “tensile strength enhancer” to the adhesive composition. Suchtensile strength enhancers preferably comprise micron-size, preferably 5to 40 microns in diameter and 20 to 5000 microns in length, high tensilestrength fibers, usually with glass transition temperatures well above37° C.

Suitable tensile strength enhancers for use in the present inventioninclude, inter alia, collagen fibers, polyglycolide and polylactidefibers, as well as other organic tensile strength enhancers andinorganic tensile strength enhancers. A particularly useful tensilestrength enhancer is VICRYL® (polyglycolide:polylactide, 90:10) Suitabletensile strength enhancers are those that have inherent high tensilestrength and also can interact by covalent or non-covalent bonds withthe polymerized gel network. The tensile strength enhancer should bondto the gel, either mechanically or covalently, in order to providetensile support. Tensile strengths of polyglycolide resorbable suturesare approximately 89,000 N/cm²; that of collagen fibers is 5000-10,000N/cm² (Hayashi, T., in Biomedical Applic. of Polym. Mater., Tsuruta, T.et al., Eds., CRC Press, Boca Raton, Fla., 1993).

The adhesive composition of the invention may also be used for localizeddelivery of various drugs and other biologically active agents inconjunction with the repair of herniated tissue. Biologically activeagents such as growth factors may be delivered from the composition to alocal tissue site in order to facilitate tissue healing andregeneration.

The term “biologically active agent” refers to an organic molecule thatexerts biological effects in vivo. Examples of biologically activeagents include, without limitation, enzymes, receptor antagonists oragonists, hormones, growth factors, autogenous bone marrow, antibiotics,antimicrobial agents and antibodies. The term “biologically activeagent” is also intended to encompass various cell types and genes thatcan be incorporated into the compositions of the invention.

Preferred biologically active agents for use in the compositions of thepresent invention are cytokines, such as transforming growth factors(TGFs), fibroblast growth factors (FGFs), platelet derived growthfactors (PDGFs), epidermal growth factors (EGFs), connective tissueactivated peptides (CTAPs), osteogenic factors, and biologically activeanalogs, fragments, and derivatives of such growth factors. Members ofthe TGF supergene family include the beta transforming growth factors(for example, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3); bone morphogeneticproteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9); heparin-binding growth factors (for example, fibroblastgrowth factor (FGF), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (forexample, Inhibin A, Inhibin B); growth differentiating factors (forexample, GDF-1); and Activins (for example, Activin A, Activin B,Activin AB). Growth factors can be isolated from native or naturalsources, such as from mammalian cells, or can be prepared synthetically,such as by recombinant DNA techniques or by various chemical processes.In addition, analogs, fragments, or derivatives of these factors can beused, provided that they exhibit at least some of the biologicalactivity of the native molecule. For example, analogs can be prepared byexpression of genes altered by site-specific mutagenesis or othergenetic engineering techniques.

Biologically active agents may be incorporated into the adhesivecomposition by admixture. Alternatively, the agents may be incorporatedinto the crosslinked polymer matrix by binding these agents to thefunctional groups on the synthetic polymers. Processes for covalentlybinding biologically active agents such as growth factors usingfunctionally activated polyethylene glycols are described in commonlyassigned U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, to Rhee et al.Such compositions preferably include linkages that can be easilybiodegraded, for example as a result of enzymatic degradation, resultingin the release of the active agent into the target tissue, where it willexert its desired therapeutic effect.

A simple method for incorporating biologically active agents containingnucleophilic groups into the crosslinked polymer composition involvesmixing the active agent with a polyelectrophilic component prior toaddition of the polynucleophilic component.

By varying the relative molar amounts of the different components of thecrosslinkable composition, it is possible to alter the net charge of theresulting crosslinked polymer composition, in order to prepare a matrixfor the delivery of a charged compound such as a protein or ionizabledrug. As such, the delivery of charged proteins or drugs, which wouldnormally diffuse rapidly out of a neutral carrier matrix, can becontrolled.

For example, if a molar excess of a polynucleophilic component is used,the resulting matrix has a net positive charge and can be used toionically bind and deliver negatively charged compounds. Examples ofnegatively charged compounds that can be delivered from these matricesinclude various drugs, cells, proteins, and polysaccharides. Negativelycharged collagens, such as succinylated collagen, and glycosaminoglycanderivatives such as sodium hyaluronate, keratan sulfate, keratosulfate,sodium chondroitin sulfate A, sodium dermatan sulfate B, sodiumchondroitin sulfate C, heparin, esterified chondroitin sulfate C, andesterified heparin, can be effectively incorporated into the crosslinkedpolymer matrix as described above.

If a molar excess of a polyelectrophilic component is used, theresulting matrix has a net negative charge and can be used to ionicallybind and deliver positively charged compounds. Examples of positivelycharged compounds that can be delivered from these matrices includevarious drugs, cells, proteins, and polysaccharides. Positively chargedcollagens, such as methylated collagen, and glycosaminoglycanderivatives such as esterified deacetylated hyaluronic acid, esterifieddeacetylated desulfated chondroitin sulfate A, esterified deacetylateddesulfated chondroitin sulfate C, deacetylated desulfated keratansulfate, deacetylated desulfated keratosulfate, esterified desulfatedheparin, and chitosan, can be effectively incorporated into thecrosslinked polymer matrix as described above.

The adhesive compositions can also be prepared to contain variouscolorants or imaging agents such as synthetic dyes and natural coloringagents, light-emissive and fluorescent dyes, iodine or barium sulfate,or fluorine, in order to aid visualization of the compositions afteradministration via optical, X-ray or ¹⁹F-MRI detection means. Suitablecolorants include, but are not limited to, FD&C dyes and FD&C lakes,(e.g., allura red AC, amaranth, brilliant blue FCF, quinoline yellow,sunset yellow FCF), black PN, Bordeaux B, Brown FK, Brown HT,canthaxanthin, carmine, carmoisine, beetroot red, chlorophyll,conchineal, curcumin, eosin, erythrosine, green S, ponceau 4R, red 2G,saffron, tartrazine, turmeric, and mixtures thereof. Examples oflight-emissive and fluorescent dyes include: fluorescein, rose bengal,indocyanine green, analogue members of the tricarbocyanine dyes; andmany others. In selecting a suitable dye, color and luminescentefficiency are two important factors. Luminescent dyes foundparticularly suitable include cyanine and related polymethine dyes,merocyanine, styryl and oxonol dyes. Other suitable coloring agents,light-emissive dyes, and fluorescent dyes will be obvious to thoseskilled in the art. It may also be desirable to incorporate proteinssuch as albumin, fibrin or fibrinogen into the crosslinked polymercomposition to promote cellular adhesion. In addition, the introductionof hydrocolloids such as carboxymethylcellulose may promote tissueadhesion.

Crosslinking of the Adhesive Composition

Any number of crosslinking techniques may be used to effect crosslinkingof the aforementioned compositions. Generally, however, components A, Band optionally C are selected such that crosslinking occurs fairlyrapidly upon admixture of all components of the crosslinkablecomposition with an aqueous medium.

For crosslinking compositions in which one or more components containhydroxyl and/or thiol groups as nucleophilic moieties, the aqueousmedium with which the crosslinking composition (or components thereof)are admixed should contain a basic reagent that is effective to increasethe nucleophilic reactivity of the hydroxyl and/or thiol group (and thusthe rate of the nucleophile-electrophile reactions) but that ispreferably non-nucleophilic so as to avoid reaction with anyelectrophilic groups present. A catalytic amount of base can be used,and/or a base-containing buffer. In an alternative but less preferredembodiment, a reactive base can be used that participates as a reactantin the crosslinking reaction.

In general, the combined concentration of all crosslinkable componentsin the aqueous admixture will be in the range of about 1 to 50 wt. %,generally about 2 to 40 wt. %. However, a preferred concentration of thecrosslinkable composition in the aqueous medium—as well as the preferredconcentration of each crosslinkable component therein—will depend on anumber of factors, including the type of component, its molecularweight, and the end use of the composition. For example, use of higherconcentrations of the crosslinkable components, or using highlyfunctionalized components, will result in the formation of a moretightly crosslinked network, producing a stiffer, more robust gel. Assuch, compositions intended for use in tissue augmentation willgenerally employ concentrations of crosslinkable components that falltoward the higher end of the preferred concentration range. Theappropriate concentration of each crosslinkable component can easily beoptimized to achieve a desired gelation time and gel strength usingroutine experimentation.

Using the adhesive composition disclosed above, peel strengths rangingfrom approximately 2 N/cm² to approximately 10 N/cm² have been observedduring in vitro testing. The test results are described in theexperimental section that follows. The peel strengths achieved weresimilar to those observed when using DERMABOND® brand (Johnson &Johnson, New Brunswick, N.J.) 2-octylcyanoacrylate, a commerciallyavailable adhesive currently used in hernia repair. Average lap shearstrengths ranging from 3.5 N/cm² to 9 N/cm² were observed in lap sheartests using BARD® mesh on cowhide. Based on these findings, were 40 cm²(4 cm×10 cm) mesh glued with the adhesive composition of the inventivemethod for hernia repair, it would require a force ranging fromapproximately 140 N (14 kg of weight force) to 360 N (36 kg of weightforce) to dislodge the entire mesh from the site.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages and modifications will beapparent to those skilled in the art to which the invention pertains.All patents, patent applications, patent publications, journal articlesand other references cited herein are incorporated by reference in theirentireties.

The following examples are included so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compounds of the invention, and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. and pressure is at or near atmospheric.

EXAMPLE 1 Comparative Peel and Lap Shear Testing

In vitro peel and lap shear tests were conducted to demonstrate theutility of the claimed adhesive materials for hernia repairapplications. Adhesive materials were applied on a 2 cm×2 cm area toglue two surfaces together. The glued surfaces were incubated inphosphate buffered saline (PBS) at 37° C. for 2 hrs, then the peelstrength and lap shear strength were measured on Instron UniversalTester, Model 4202, Canton Mass.

Materials

The adhesive compositions were tested on the following materials:

-   -   Polypropylene mesh: Lab grade and BARD® Mesh, Davol, Inc.        Cranston, R.I.    -   Collagen membrane: Collagen sausage casing #1, 2, and 3, The        SausageMaker, Inc., Buffalo, N.Y.    -   Cowhide strip: fresh cowhide strip from Spear Products, Inc.,        Quakertown, Pa.

The adhesive compositions were as follows.

Composition A:

-   -   Component I        -   100 mg pentaerythritol polyethylene glycol ether            tetra-succinimidyl glutarate, (MW, 10K); Shearwater            Polymers, Huntsville, Ala. and        -   100 mg pentaerythritol polyethylene glycol ether            tetra-sulfhydryl Shearwater Polymers, (MW, 10K), Huntsville,            Ala., in a syringe.    -   Component II        -   1 ml of 20 mg/ml methylated collagen in a separate but            connected syringe.

Methylated collagen was prepared by a modification of the procedure ofMiyata et al, U.S. Pat. No. 4,164,559. A dispersion (3% w/v) of bovinepepsinized reconstituted collagen in 0.02M sodium phosphate, 0.13M NaCl,pH 7.2 (prepared by the method of McPherson et al., Collagen Rel. Res.5, 119-135, 1985) was extruded onto a nonsticky glass surface in a thinstring and dried at room temperature. Methanolic HCl was prepared byadding 10.7 ml of conc. HCl to 1300 ml of anhydrous methanol. The driedcollagen was cut into 1×5 cm strips and added to the methanolic HCl (200ml methanolic HCl: 1 g dry collagen) in a sealed vessel and gentlyshaken at 25° C. for 3 days. The methanolic HCl was carefully decantedoff and the collagen was filtered on a sintered glass funnel to removetraces of methanol. Complete methanol and acid removal was completedunder vacuum overnight or dialysis against H₂O extensively. Themethylated collagen was re-solubilized in 20 mM acetate buffer, and thepH was adjusted to 4 to 6. The amount of buffer was calculated toachieve a final protein concentration of about 20 mg/ml. Solubilizedmethylated collagen was a completely transparent material, free offibers or opalescence, having a viscous, gel-like consistency.

Components I and II were thoroughly mixed between the two syringesthrough the connector, and this composition was then applied to thesurface of the test material and spread evenly to a thickness of 0.1 mlof the mixture per cm² area to be glued. A pH 9.6 NaH₂PO₄/Na₂CO₃ bufferwas applied drop-wise to the Composition A coating on the mesh. Afterallowing the coated, buffered mesh to stand at room temperature for 20minutes, the mesh coating was rinsed thoroughly with PBS to remove thebuffer.

Composition B:

-   -   Component I        -   100 mg pentaerythritol polyethylene glycol ether            tetra-succinimidyl glutarate, and        -   100 mg pentaerythritol polyethylene glycol ether            tetra-sulfhydryl, in one syringe.    -   Component II        -   1 ml of 20 mg/ml methylated collagen prepared as discussed            above in another syringe.    -   Component III        -   38 mg poly(L-lactic acid) fiber (PLLA) Transome, Inc. Palm            Bay, Fla., washed with isopropyl alcohol and treated with            H₂O₂.

Components I, II, and III were thoroughly mixed in a beaker, 0.1 ml ofthe mixture per cm² area to be glued was applied and then spread evenlyon the surface of the test material. A pH 9.6 NaH₂PO₄/Na₂CO₂ buffer wasapplied drop-wise on top of the coated surface to cover the coatedsurface. After allowing the coated, buffered surface to stand at roomtemperature for 20 min, the coated test material was rinse thoroughlywith PBS to remove the buffer.

DERMABOND®: 2-Octylcyanoacrylate

TISSEEL®: fibrin glue.

KRAZY GLUE®: Ethyl Cyanoacrylate.

Methylated Collagen: 20 mg/ml prepared as discussed above.

Pentaerythritol Polyethylene Glycol Ether Tetra-SuccinimidylGlutarate/Pentaerythritol Polyethylene Glycol Ether Tetra-Sulfhydryl:100 mg/l 00 mg in 1 ml.

ZYDERM® Collagen Implant II: 65 mg/ml fibrillar collagen in PBS.

EXAMPLE 2 Comparative Peel Strength in 2×2 cm PolypropyleneMesh/Collagen Membrane Testing

The surface characteristics of the materials used in the test modelrequire standardization to give consistent results. Four types ofcollagen membrane surfaces gave different peel strength results with labgrade polypropylene mesh for the same Composition A formulation. Thedata are shown in FIG. 11. The affixed mesh/collagen membranes wereincubated for 2 hours at 37° C. in PBS.

EXAMPLE 3 Comparative Peel Strength in 2×2 cm PolypropyleneMesh/Collagen Membrane Testing

The peel strength for the various adhesives when used to adhere labgrade polypropylene mesh to a collagen membrane surface was determined.The amount of force required to peel the lab grade polypropylene meshoff of the membrane surface glued by different materials afterincubation in PBS for 2 hrs at 37° C. varied from zero to ˜0.35 N/cmlinear width. Composition A and Composition B gave the same peelstrength as KRAZY GLUE® and DEREMABOND®, cyanoacrylate products.TISSEEL® gave lower peel strength, <0.1 N/cm lineal width. The data areshown in FIG. 12.

EXAMPLE 4 Comparative Peel Strength for Various Incubation Times forCompound A in 2×2 cm Polypropylene Mesh/Cowhide Strip Testing

Changes in the peel strength of Composition A when used to affixpolypropylene mesh to a cowhide strip did not change significantly after24 hrs incubation in PBS at 37° C. The data are shown in FIG. 13.

EXAMPLE 5 Comparative Lap Shear Strength for Various Incubation Timesfor Compound A in 2×2 cm Polypropylene Mesh/Cowhide Strip Testing:

The lap shear strength of Composition A, Composition B, KRAZY GLUE® andTISSEEL® when used to affix a 2×2 cm polypropylene mesh strip onto acowhide strip. The average pull strength for Composition A andComposition B was 2 N/cm² and 4.5 N/cm² respectively in a lab gradepolypropylene mesh on cowhide strip model. The average lap shearstrength of Composition B is statistically significantly higher thanKRAZY GLUE® and TISSEEL®'s average lap shear strength, 1.3 N/cm² in thismodel. The average lap shear strength of Composition A is also higherthan KRAZY GLUE® and TISSEEL®'s average lap shear strength of 1.3 N/cm²,but it is not statistically significantly in this model. The data areshown in FIG. 14.

EXAMPLE 6 Comparative Lap Shear Strength for Compound A and Compound Bin 2×2 cm Lab Polypropylene Mesh or BARD® Mesh/Cowhide Strip Testing

The lap shear strength of Composition A and Composition B when used toaffix lab grad polypropylene mesh and BARD® polypropylene mesh oncowhide strip was determined. The lap shear strength of Composition Aincreased from ˜2 N/cm² when used with lab. grade mesh to ˜3.5 N/cm²when used with BARD® mesh. Similarly, the pull strength of Composition Bincreased from ˜4.5 N/cm² with lab. grade mesh to ˜9.0 N/cm² with BARD®Mesh. The data are shown in FIG. 15. As Composition B containsadditional PLLA fibers, interaction of the fibers with surfaces havinginterlocking mechanisms such as the BARD® Mesh served to increaseincreased lap shear strength.

EXAMPLE 7 Pentaerythritol Polyethylene Glycol Ether Tetra-SuccinimidylGlutarate/Pentaerythritol Polyethylene Glycol EtherTetra-Sulfhydryl/Methylated Collagen Plus The Fibrous Fillers Glass Woolor VICRYL®

Materials:

Methylated Collagen, Prepared As Described in Example 1:

Adhesive Without Filler:

For 0.5 ml of adhesive, 50 mg of dry powdered pentaerythritolpolyethylene glycol ether tetra-succinimidyl glutarate and 50 mg of drypowdered pentaerythritol polyethylene glycol ether tetra-sulfhydryl weremixed with 400 mg of methylated collagen at 31 mg protein/ml, pH 4. BothPEG components dissolved in the aqueous solution of collagen, yielding atransparent, viscous fluid. The solution was spread on the tissue sitewith a spatula; it flowed very little under the force of gravity. Tocure the adhesive, 20-50 μl of a buffer (either 134 mM sodium phosphate,166 mM sodium carbonate, pH 8.9; or NaH₂PO₄/Na₂CO₃ buffer, pH 9.6) wasadded to the surface. The buffer did not dilute the gel, but slowlysoaked in. In 3-5 min, the surface of the gel was noticeably hardened.

For studies of bond strength under hydrated conditions, the gel plussubstrate was allowed to cure for 20 min on the bench, then immersed in50 mM sodium phosphate, 130 mM sodium chloride, pH 6.7, at 37° C. for 2hours or longer. Testing of bond strength was performed on a tensileapparatus.

Adhesive with Filler:

VICRYL® is a copolymer of glycolic acid and lactic acid (90:10) sold asan implantable mesh by Ethicon Corporation (Polyglactin 910;Sommerville, N.J.). To the methylated collagen was added 19 mg ofVICRYL® threads 1-2 cm long which had been unraveled from implantableVICRYL® mesh. In some cases, VICRYL® fibers as short as 0.3 cm were alsoused. The threads and the viscous gel were blended, and then the PEGcomponents were added, as described above. Application to the tissuesite and curing were as above. Other fillers and their respectiveamounts added to 0.5 ml of adhesive were: glass wool, 9 mg; fibrouscollagen (Semed F collagen, Kensey-Nash Corporation) 8 mg; Dexon S (polyglycolide lactide sutures, “4-0”), 10 pieces 1 cm long; elastin fibers(bovine neck ligament, 0.25 to 10 mm, Elastin Products Co., Inc,Owensville, Mo.), 40 mg; stainless steel fibers (Bekaert FibreTechnologies, Marietta, Ga.), 14-28 mg (Fibers were washed with water or1N HCl to remove a polyvinylalcohol coating); polylactide/glycolidemicro-particles, prepared from polylactide/glycolide (65:35, 40-75,000mol. wt., Aldrich Chemical Co., micro-particles 2-4 diameter prepared bythe method of Zheng, J., and Hornsby, P. J., Biotechnol. Progr. 15,763-767 (1999), 25 mg.

Adhesive with Methylated Collagen Replaced by Another Agent:

Various long-chain molecules were tested, such as hyaluronic acid(rooster comb, Sigma Chemical Co., St. Louis, Mo.), chitosan (Sigma),and polylysine (Sigma). For hyaluronic acid, the formula was:pentaerythritol polyethylene glycol ether tetra-succinimidyl glutarate,50 mg, pentaerythritol polyethylene glycol ether tetra-sulfhydryl, 50mg, VICRYL®, 14 mg, and 400 μl of hyaluronic acid, 2% (w/v) in water, pHadjusted to 4; for chitosan, the same formula, with 400 μl of 1%chitosan (w/v) in water, pH 4-5. For polylysine, pentaerythritolpolyethylene glycol ether tetra-succinimidyl glutarate, 40 mg,pentaerythritol polyethylene glycol ether tetra-sulfhydryl, 30 mg,dissolved together in 50 μl water; polylysine hydrobromide, 330 K, 40 mgdissolved in 6011 water; the two solutions were mixed together, and 7 mgVICRYL® fibrils were added. In addition, polylactide/glycolideparticles, prepared as above, were tested as a replacement formethylated collagen; 16.5 mg of particles were suspended in 300 μl ofwater and mixed with 50 mg pentaerythritol polyethylene glycol ethertetra-succinimidyl glutarate, 50 mg pentaerythritol polyethylene glycolether tetra-sulfhydryl, and 14 mg VICRYL®. All gels were cured with pH9.6 buffer overlay, as described above.

Adhesive Without Filler and Without Methylated Collagen:

Pentaerythritol polyethylene glycol ether tetra-succinimidyl glutaratewas dissolved in water at 20% (w/v); pentaerythritol polyethylene glycolether tetra-sulfhydryl was dissolved at 20% in pH 8.9 buffer. The twosolutions were rapidly mixed and extruded onto the site. Gelationoccurred in ˜40 sec.

Mechanical Tests:

Bond strength of the adhesive formulations were determined for each ofthe composition when applied to three types of tissue or tissuesurrogates. Collagen membranes (sausage casings; The SausageMaker, Inc.,Buffalo, N.Y.) were washed with isopropyl alcohol and water to removelipid and salt impurities, and dried. Bonding of membranes with a 1-3 mmoverlap and a 1 cm width was performed by spreading the adhesive overthe top of the sheets. Adhesive was allowed to cure 20 min on the benchand then immersed for 30 min to 2 hours at 37° C. before pulling apartin an Instron model 4202 test apparatus (Canton, Mass.), using a 100 Nload cell. Bonding of porcine carotid arteries (10 b, Pelfreeze, Rogers,Ark.) was also performed in an end-to-end geometry. Cut carotid arterysegments were abutted (4-6 mm diameter) and spread with adhesive; nostay sutures were applied. Incubation and testing were the same asdescribed for the collagen membranes.

For bonding of cowhide strips (10 c), de-haired calfskin pieces werepurchased from Spear Products, Inc., Quakertown, Pa. Pieces were nearlyuniform in thickness, 2-3 mm. Strips 0.4 cm wide were cut from the hidepieces, using a single-edged razor blade. Cut strips were abuttedend-to-end and bonded by spreading 0.25 ml of Composition A adhesive ora few drops of cyanoacrylate. Incubation and testing were the same asdescribed for the collagen membranes. Table 3 below shows thatpentaerythritol polyethylene glycol ether tetra-succinimidylglutarate/pentaerythritol polyethylene glycol ethertetra-sulfhydryl/methylated collagen, when filled with glass wool(Formula c), was superior in bonding strength to unfilled Formulas a andb when tested on collagen membranes. A medical grade cyanoacrylate(DERMABOND®) formed even stronger bonds with collagen membranes (5.2±1.9N force for 7 determinations).

TABLE 3 Bonding Performance With And Without Methylated Collagen And AFibrous Filler Bond Strength Formula (N Force) n PentaerythritolPolyethylene Glycol Ether Tetra-   1.6 ± 1.1 3 SuccinimidylGlutarate/Pentaerythritol Polyethylene Glycol Ether Tetra-Sulfhydryl(20%) Pentaerythritol Polyethylene Glycol Ether Tetra-   1.7 ± 1.0 4Succininidyl Glutarate/Pentaerythritol Polyethylene Glycol EtherTetra-Sulfhydryl/methylated collagen Pentaerythritol polyethylene GlycolEther Tetra- >2.8 ± 0.6* 6 Succinimidyl Glutarate/PentaerythritolPolyethylene Glycol Ether Tetra-Sulfhydryl/methylated collagen/glasswool *Collagen membrane tore, but sealant bond was still intact.

TABLE 4 Bond Strength of Cyanoacrylate (KRAZY GLUE ®, Elmer's Products)on Three Different Tissue Substrates Substrate Bond Strength (N Force)Cowhide strips 10.9, 16.2 Porcine carotid artery 2.0, 3.8 Collagenmembrane 3.0 ± 1.0 (n = 5)

Table 5 below presents data on the addition of a different filler,VICRYL® threads, to the pentaerythritol polyethylene glycol ethertetra-succinimidyl glutarate/pentaerythritol polyethylene glycol ethertetra-sulfhydryl/methylated collagen. With substrates such as cowhide orcarotid artery, the substrate did not tear, and the bond strength valueswere representative for the strength of the adhesive bond itself.Typically these bonds failed adhesively, that is, the tensile strengthof the adhesive gel itself remained intact and was not the limitingfactor. The bond strengths observed in Saline at 37° C. again werecomparable to those seen with cyanoacrylate for bonding the same set oftissue substrates (Table 4).

TABLE 5 Bond Strength of Pentaerythritol Polyethylene Glycol EtherTetra-Succinimidyl Glutarate/Pentaerythritol Polyethylene Glycol EtherTetra-Sulfhydryl/Methylated Collagen with VICRYL ® Threads as a Filleron Three Different Tissue Substrates Incubation Bond Strength Time(Hrs.) (N Force) Substrate* 2 6.6, 5.6 Cowhide 17 6.3, 5.5 Cowhide 24.3, 2.2, Porcine 2.8, 5.1 Carotid Artery 2 >5.9, 3.9 Collagen Membrane*cowhide strips, 0.5 cm wide, porcine carotid artery, 0.3-0.5 cmdiameter, collagen membrane: sausage casing, 0.2 mm thick, 1 cm width.Effect of Different Fillers:

Table 6 presents results of various filler materials. Testing wasperformed on cowhide strips, immersed for 2 hours in saline at 37° C. Itappeared that filamentous materials were more effective than spheroidalparticles. Bonding of the filler to the gel is very important forimprovement of strength. Collagen-polyethylene glycol filaments werewaxy and did not adhere to the gel; thus, despite their high aspectratios, they were not effective fillers.

TABLE 6 Effect of Different Fillers on Bond Strength of PentaerythritolPolyethylene Glycol Ether Tetra-Succinimidyl Glutarate/ PentaerythritolPolyethylene Glycol Ether Tetra-Sulfhydryl/Methylated Collagen BondStrength Material (N Force) VICRYL ® 4.7, 7.4 VICRYL ®, washed withethanol 7.2, 7.8 VICRYL ®, treated with ethanol, then washed with 8.3,9.1 30% hydrogen peroxide Surgical silk sutures 1-2 cm long, 30-50μdiameter 2.5, 3.8 Surgical silk sutures, unraveled to finer threads,5.0, 6.5 washed with chloroform Fibrous collagen (Semed F, Kensy-Nash)adjusted to 1.3, 2.8 pH 4; 0.5 to 1 mm long, ~50μ diameter Gelatinparticles, cross-linked by heat, ~100μ 0.6, 0.8 diameter, polygonalHydroxyapatite particles, 0.5 to 1 mm diam. 0.7 polygonalCollagen-polyethylene glycol conjugate filament 0.8, 1.7 ~50 μ diameter,1 cm long Stainless steel fibers 8 μ diameter, 4 mm long 4.8, 6.9Elastin fibers 0.25 to 10 mm long 3.9, 4.0 Polylactide/glycolideparticles, 2-4μ diameter 1.1, 1.1Effect of Cross-Linking Bond:

Table 7 below shows that when the gel was formed from other types ofcross-linking reactions, the adhesion and bond strength was affectedwhen tested on cowhides after incubation at 37° C. Material 1 was formedfrom pentaerythritol polyethylene glycol ether tetra-sulthydryl andhydrogen peroxide, which oxidizes adjacent sulfhydryl groups to adisulfide bond. A gel forms rapidly, and the gel can be supplementedwith methylated collagen and VICRYL® (Johnson & Johnson, New Brunswick,N.J.); however, after several hours in saline buffer, the gel becomesvery weak; the VICRYL® fibers are easily pulled out. Material 2 utilizedthe reaction of sulfhydryl groups from pentaerythritol polyethyleneglycol ether tetra-sulfhydryl with the double bond of a 4-arm vinylsulfone derivative of PEG (10K, Shearwater Polymers). The presumedreaction, a Michael-type addition, formed a thio-ether bond. Such gelshad adequate tensile strength but poor adhesion to the cowhide afterincubation in saline. Materials 3 and 4 contained (pentaerythritolpolyethylene glycol ether tetra-amine, 10K, Shearwater Polymers); theamino functionality presumably reacted with the succinimidyl ester ofpentaerythritol polyethylene glycol ether tetra-succinimidyl glutarateto form an amide linkage. These gels were comparable in performance tothose formed from pentaerythritol polyethylene glycol ethertetra-succinimidyl glutarate and pentaerythritol polyethylene glycolether tetra-sulfhydryl. (For proper reaction in the presence ofmethylated collagen, the pentaerythritol polyethylene glycol ethertetra-amine had to be titrated to pH 2-4 during the mixing of reagents;on addition of curing buffer, its pH was increased, permitting thereaction of the amino group). It appeared that the presence of thesuccinimidyl ester was important for achieving the highest adhesion tothe tissue substrate and for good tensile strength of the gel. Othergroups that react with amines, such as aldehydes (aldehydes conjugatedto multi-armed PEG), are also anticipated to be effectiveadhesive-forming reagents.

TABLE 7 Bond Strengths of Various Functionalized PEGs Filled withVICRYL ® Threads Material Incubation Bond No. Material Time (Hrs.) (NForce) 1 Pentaerythritol Polyethylene 17 0.32, 0.20 Glycol EtherTetra-Sulfhydryl/ Methylated Collagen/Vicryl/H₂0₂ 2 PentaerythritolPolyethylene 2 2.2, 1.5 Glycol Ether Tetra-Sulfhydryl/ 4arm vinyl 2sulfone PEG/Metylated Collagen/Vicryl threads 3 PentaerythritolPolyethylene 2 6.4 Glycol Ether Tetra-SuccinimidylGlutarate/Pentaerythritol Polyethylene Glycol Ether Tetra-Sulfhydryl/Pentaerythritol Polyethylene Glycol Ether Tetra-Amine/Methylated Collagen/Vicryl threads 4 Pentaerythritol Polyethylene4 3.6, 6.4 Glycol Ether Tetra-Succinimidyl Glutarate/PentaerythritolPolyethylene Glycol Ether Tetra- Amine/Methylated collagen/Vicrylthreads 4 Pentaerythritol Polyethylene 2 6.6, 5.6 Glycol EtherTetra-Succinimidyl Glutarate/Pentaerythritol Polyethylene Glycol EtherTetra- Amine/Methylated collagen/Vicryl threadsPersistence of the Bond Under Hydrated Conditions:

Table 8 shows that the adhesives formed from pentaerythritolpolyethylene glycol ether tetra-succinimidyl glutarate, pentaerythritolpolyethylene glycol ether tetra-sulfhydryl, and also pentaerythritolpolyethylene glycol ether tetra-amine form bonds using cowhide thatpersist for long times immersed in saline buffer at 37° C. Suchstringent hydrated conditions simulate the in vivo environment. Bondweakening was observed after more than 100 hours of hydration. Theweakening of bond strength was thought to be due to hydrolysis ofcarboxyl-ester and thio-ester network linkages. Pentaerythritolpolyethylene glycol ether tetra-succinimidyl glutarate is aglutaryl-succinimidyl ester; even after reaction with the terminalcarboxyl of the succinimidyl ester, there remains a carboxyl esterlinking the glutaryl moiety to the main PEG chain; this bond, as well asthe thio-ester bond, could hydrolyze.

TABLE 8 Bond Performance Under Long Hydration Times Incubation Time BondStrength (N Material (Hrs.) Force) Pentaerythritol Polyethylene GlycolEther 2 6.4 Tetra-Succinimidyl 66 2.6, 4.1 Glutarate/PentaerythritolPolyethylene 70 3.0 Glycol Ether Tetra- 137 0.70, 2.6Sulfhydryl/Pentaerythritol Polyethylene Glycol Ether Tetra-Amine/ 1401.1, 0.4 Methylated collagen/ VICRYL ® threads PentaerythritolPolyethylene Glycol Ether 4 3.6, 6.4 Tetra-Succinimidyl 64 7.0, 5.1Glutarate/Pentaerythritol Polyethylene 136 3.8, 2.7 Glycol EtherTetra-Amine/ 234 2.7, 1.7 Methylated collagen/ VICRYL ® threadsPentaerythritol Polyethylene Glycol Ether 2 6.6, 5.6 Tetra-SuccinimidylGlutarate/ 17 6.3, 5.5 Pentaerythritol Polyethylene Glycol EtherTetra-Sulfhydryl/ 69 0.63, 0.90, 3.4, 5.4 Methylated collagen/ 93 2.4,5.4 VICRYL ® threads 140 3.2, 2.9 235 >2.4, 3.7Related Formulas with Lower Molecular Weight Compounds BearingSuccinimidyl Ester and Amino or Thiol Reactive Groups:

Table 9 presents bond strengths on cowhide strips of lower molecularweight PEG derivatives as adhesives, again supplemented with methylatedcollagen and VICRYL®. Tri-functional succinimidyl-succinate of a 3-armedPEG built from a glycerol core, 2600 mol. wt., was obtained from NOFCorporation, Japan and 4-armed polyethylene glycol di-amine, 2000 mol.wt., was obtained from Shearwater Polymers. The polymers were VICRYL®filling appeared to have a small effect on bond strength. The followingproportions were used: methylated collagen, 500 μl (22 mg/ml in water2707-30B); tri-functional succinimidyl-succinate of a 3-armed PEG builtfrom a glycerol core, 48 mg; 4-armed polyethylene glycol di-amine, 2000mol. wt., Shearwater Polymers, 60 μl of 60% solution in water, titratedto pH 1-2 with 6M HCl; VICRYL® threads, 26 mg.

TABLE 9 Low Molecular Weight Analogues to Pentaerythritol PolyethyleneGlycol Ether Tetra-Succinimidyl Glutarate and PentaerythritolPolyethylene Glycol Ether Tetra-Sulfhydryl Incubation Bond Time StrengthMaterials (Hrs.) (N Force) Tri-functional succinimidyl-succinate of a 3-2 2.3, 0.64 armed PEG built from a glycerol core/ 4-Armed PolyethyleneGlycol Di-Amine/ Methyated Collagen Tri-functionalsuccinimidyl-succinate of a 3- 5 2.3, 3.3 armed PEG built from aglycerol core/ 4-armed polyethylene glycol di-amine, 2000 mol. wt.,Shearwater Polymers/Methylated Collagen/VICRYL ® threads

EXAMPLE 8 Lap Shear Testing of the Adhesive of the Invention vs.TISSUCOL®

The mechanical strength of an adhesive of the invention and TISSUCOL®(Immuno Aktiengesellschaft, Wien, Austria) were evaluated using asurvival rat flap model. Composition A, as described in Example 1, wasused as the adhesive and delivered with a CoStasis cannula. Data from 14animals is reported: 7 rats per the adhesive group and 7 rats perTISSUCOL® group. A 2×3 cm skin flap was made on the chest of each rat.The lap shear force (N/cm2) required to detach the mesh was measured.The mean force for the adhesive of the invention at 3 days was 1.1±0.06N/cm2. The mean peel force for TISSUCOL® at 3 days was 1.57+0.54 N/cm2.Mechanical test results were evaluated by non-parametricWilcoxon/Kruskal-Wallis analysis and Tukey-Kramer HSD parametricanalyses to determine p-values for significance. Composition A andTISSUCOL® were not statistically significantly different. The data fromthis study demonstrate that Composition A is mechanically as strong asTISSUCOL®.

Methods:

This study included a total of 14 rats, divided into four treatmentgroups. A 2×3 cm skin flap was made on the chest of a rat. Once the skinflap was raised, 0.4 mL of Composition A was placed onto the muscleusing a cannula. The mesh was laid down over the material and counterbuffer was sprayed on to initiate crosslinking of the adhesive. Thetissue closed after 2 minutes using staples in the same manner for allgroups. The animals were euthanized 3 days after application. Thestaples used for closure of the surgical site were removed formechanical testing. For mechanical testing, the euthanized rats weresecured onto a board with straps. The end of the mesh was raised andplaced in a clip attached to the upper jig of the Instron, Model 4202Serial #246 (Collagen Corp Asset #1225). The Instron was setup formeasuring tension. The speed was set at 10 mm/min using a 100 NewtonLoad Cell. The grip pulls up on the skin separating it from the muscle.The mesh pulls apart from the muscle leaving a thin layer of theadhesive on both the mesh side and the muscle side.

Statistical Method:

The average and standard deviations (SD) for peel strength (N/cm) foreach group were determined using raw data. Mean peel strengths wereevaluated by nonparametric Wilcoxon/Kruskal-Wallis Tests (Rank Sums) andTukey-Kramer HSD parametric analyses to determine p-values forsignificance. All analyses were performed using JMP statistical package,version 3.0 (SAS Institute).

Results:

The biological response to Composition A and TISSUCOL® was evaluated.Overall, Composition A and TISSUCOL® as well as the hernia mesh seemedbiocompatible at 3-days post implantation. Each of the adhesives flowedinto the mesh spaces and could be seen histologically at 3 days.

The strength of attachment was measured at three days post implantation.Composition A showed a lap shear of 1.11 N/cm² with a standard deviationof 0.06, while the TISSUCOL® demonstrated a lap shear of 1.57 N/cm witha standard deviation of 0.54. Although the mean for TISSUCOL® is higherthan that of the adhesive of the invention, there is not a statisticallysignificant difference between the two groups. Composition A was notstatistically significantly stronger than TISSUCOL®, p≧0.05(Tukey-Kramer). Also, adhesions, mesh to skin, were found in every casewith TISSUCOL® whereas Composition A had no adhesions.

EXAMPLE 9 Lap Shear Testing of Hernia Mesh Attached with Composition Aand TISSUCOL® In Vivo

Rats were implanted in the ventral thoracic region with hernia meshcoated with either Composition A or TISSUCOL®. Animals were sacrificed3-days post operative and the implants subjected to mechanical testingand/or prepared for microscopic evaluation. Both of the composites werebiocompatible showing only a few macrophages on the surface of theattachment materials and in surrounding subcutaneous tissues.Polymorphonuclear neutrophils penetrated TISSUCOL® making it appearporous however; Composition A was generally acellular. The latterproperties of the two materials could affect their individual turnoverrates and the time course of tissue in growth necessary for hernia meshfixation.

Materials and Methods:

Eleven rats were implanted in the mid-thoracic region with a compositeof either hernia mesh/Composition A or a hernia mesh/TISSUCOL®. Theimplants and surrounding tissues were harvested at 3-days postimplantation and processed for paraffin and glycolmethacrylate (GMA)plastic embedding either before or after mechanical testing. In mostcases, the composite implants and the surrounding tissues were embeddedin separate blocks. Paraffin sections were stained with hematoxylin andeosin (H & E) and Masson's trichrome stain; plastic sections werestained with H & E only. A total of 50 slides from the sites wereevaluated microscopically for presence, site and cellularity of theimplant materials. The appearance of serum pockets and types ofinflammatory cells were also noted. Observations were recorded and usedas the basis for this text.

Results:

There was no tissue reaction in the skin above the implant sites. Low tomoderate numbers of macrophages was present in the subcutaneous tissuesassociated with both the hernia mesh/Composition A and the herniamesh/TISSUCOL® composites. Serum pockets containing fibrin and bloodwere also seen in the subcutaneous layer. These probably resulted fromcreating and elevating the skin flaps for placements of the implants.

After mechanical testing, both Composition A and TISSUCOL® stayed withthe mesh with little, if any, material remaining attached to the hosttissues. A small amount of tissue was sometimes associated with theTISSUCOL®/mesh complex, but no tissue pulled way with the CompositionA/mesh complex after testing. The attachment materials filled the voidspaces or pores of the mesh, but there was no indication of eithermaterial or cellular penetration of the weave of the mesh. When the meshwas viewed with polarized light a layer of Composition A sometimescovered it, while the mesh was usually completely exposed in sections ofTISSUCOL®. This might suggest that a thicker layer of Composition A wasapplied to the mesh compared to the amount of TISSUCOL® used for coatingthe mesh.

Composition A was completely acellular and showed no evidence ofdegradation at this early time point. This material elicited minimaltissue reaction with only a few macrophages and polymorphonnuclearneutrophils (PMN's or neutrophils) associated with its surface. A fewmacrophages were also seen on the surface of TISSUCOL® however, a largenumbers of polymorphonuclear neutrophils (PMN's) were observedsurrounding this material. Some of the PMN's had penetrated the surfaceof the TISSUCOL® giving it a spongy, porous appearance.

There was no indication that the mesh was responsible for any of thecellular responses associated with the attachment materials or seen inthe surrounding subcutaneous tissues. However, implants of the meshalone would have provided a definitive answer to this question and alsoestablished baseline mechanical testing data.

The large numbers of PMN's associated with TISSUCOL® may not be causefor concern since the material contains fibrinogen and fibrinogenfragments can act as chemo attractants for these cells during normalwound healing. Also, PMN's are often the first to appear at a site ofsoft tissue injury as well as following placement of an implant.Generally, if they do not persist beyond a week or two there is littlecause for concern. If they persist beyond this time period then somechronic irritant on infection is likely present. Other than the PMNinfiltrate associated with TISSUCOL®, tissue reactivity was mild. Therewas a slight increase in the number of tissue macrophages with bothattachment materials. The sequential appearance of increased numbers ofmacrophages after PMN's peak also occurs during normal wound healing.Hence, biocompatibility does not seem to be a problem with eithermaterial at this time point.

The presence of holes in TISSUCOL® after cell penetration may suggestthat it will turnover faster than Composition A that shows no cellinfiltration or evidence of degradation at this early time point. Theissue is whether a faster or slower turnover rate for attachmentmaterials is more advantageous in this clinical indication. If the mostimportant function of the attachment material is initial stabilizationand the most desirable final fixation of the mesh is tissue in growth,then a material that turns over faster might be more desirable. Fasterturnover might favor faster in growth and tissue fixation while anattachment material with a higher persistence might hinder penetrationof host tissue.

1. A mesh for use in the repair of herniated tissue comprising a meshand a coating composition including: (a) a naturally occurringhydrophilic polymer selected from proteins, polysaccharides, andglycosaminoglycans, wherein the naturally occurring hydrophilic polymeris not methylated collagen; (b) a first synthetic polymer having mnucleophilic groups, wherein m≧2; (c) a second synthetic polymer havingn electrophilic groups capable of reaction with the m nucleophilicgroups to form covalent bonds, wherein n≧2; and wherein each of thefirst synthetic polymer and the second synthetic polymer is poly(alkylene oxide) and crosslinking of the coating composition results ina biocompatible, crosslinked matrix.
 2. The mesh of claim 1, wherein thefirst synthetic polymer and the second synthetic polymer arehydrophilic.
 3. The mesh of claim 1, wherein m+n is 5 or more.
 4. Themesh of claim 1, wherein each of the first synthetic polymer and thesecond synthetic polymer is nonimmunogenic.
 5. The mesh of claim 1,wherein the naturally occurring hydrophilic polymer is selected fromnonfibrillar collagen, succinylated collagen, fibrillar collagen,denatured collagen and combinations thereof.
 6. The mesh of claim 5,wherein the nonfibrillar collagen is selected from type IV collagen,type VI collagen, and type VII collagen.
 7. The mesh of claim 2, whereinthe first synthetic polymer and the second synthetic polymer arepolyalkylene oxides.
 8. The mesh of claim 7, wherein the first syntheticpolymer and the second synthetic polymer are polyethylene glycol.
 9. Themesh of claim 1, wherein the nucleophilic groups of the first syntheticpolymer can be the same or different and are —NH₂, —SH, —OH, —PH₂, or—CO—NH—NH₂.
 10. The mesh of claim 1, wherein the coating compositionfurther comprises a biologically active agent.
 11. The mesh of claim 1,wherein the electrophilic groups are a succinimidyl ester group, asuccinimidyl carbonate group, and a maleimidyl group, —COOH, —CHO,—N═C═O or —SO₂CH—CH₂.